Recombinant antigen for detecting HIV antibody

Abstract

The present invention relates to the technical field of immunodiagnosis, and provides a recombinant HIV antigen for detecting an HIV antibody. The present invention specifically relates to a recombinant HIV antigen, a nucleic acid molecule, an expression vector, a host cell, a reagent, a kit, and a use. Using a recombinant HIV antigen protein for a marking end can remarkably improve the activity of a marker of a recombinant HIV antigen, can reduce the non-specific immune binding activity, and can improve the sensitivity.

Description

Recombinant antigens for detecting HIV antibodies

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 2023107487351, filed with the Chinese Patent Office on June 21, 2023, entitled “Recombinant Antigen for Detecting HIV Antibodies,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure belongs to the technical field of immunodiagnosis and relates to a recombinant antigen for detecting HIV antibodies. Background Art

[0004] The following statements merely provide background information related to the present disclosure and may not necessarily constitute prior art.

[0005] There are currently two types of HIV (human immunodeficiency virus): HIV-1 and HIV-2. HIV-1 is currently the main cause of ADIS. Based on different cross-species transmission chains, HIV-1 can be divided into: M type, N type, O type, and P type. Among them, M type accounts for 90% of HIV-1 virus infections. Based on different epidemic areas, M type can be divided into subtypes A, B, C, D, E, F, G, H, I, J, K, and L. HIV-2 has weaker transmission ability and toxicity than HIV-1. There are currently 8 known types of HIV-2, only 2 of which have caused large-scale epidemics, namely group A and group B.

[0006] HIV is a spherical particle with a diameter of 100 to 120 nm, composed of a core and an envelope. The inner region consists of a conical core surrounded by the structural protein p24 (capsid). The core is composed of the ssRNA genome (enclosed by the p7 nucleocapsid), reverse transcriptase, integrase, and protease, along with several minor proteins and the primary core protein. A limited number of HIV envelope glycoproteins (Env) are found on the virion's surface and are responsible for binding to its primary host receptor, CD4, and its co-receptors (primarily CCR5 or CXCR4), leading to viral entry into target cells. The HIV-2 env gene encodes the gp160 precursor protein, which is cleaved into gp125 and gp36 proteins by the host cell protease furin within the host cell.

[0007] HIV antibody testing is one of the main methods for screening HIV-infected patients. gp36 protein is the main antigen raw material for HIV-2 antibody testing. However, HIV gp36 is very hydrophobic, and it is usually difficult to prepare products with good detection performance.

[0008] Application Contents

[0009] The present disclosure proposes a gp36 protein sequence as shown in SEQ NO. 1 for detecting HIV-2 antibodies.

[0010] In some embodiments of the present disclosure, a portion of the gp36 protein sequence shown in SEQ NO. 1 is used to detect HIV-2 antibodies, such as the segment from amino acid position x to amino acid position y of the gp36 protein, where x is any integer from 1 to 60, and y is any integer from 143 to 161.

[0011] 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, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60; y is 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161.

[0012] In some embodiments of the present disclosure, the HIV recombinant antigen comprises a segment of amino acids 1-143 of gp36 as shown in SEQ NO.2, a segment of amino acids 15-143 as shown in SEQ NO.3, or a segment of amino acids 60-161 as shown in SEQ NO.4.

[0013] In some embodiments of the present disclosure, the cysteine ​​in the recombinant antigen is mutated to other amino acids. In an optional embodiment, the cysteine ​​is mutated to serine.

[0014] In some embodiments of the present disclosure, the recombinant antigen further comprises segments of other HIV proteins. In an optional embodiment, the recombinant antigen further comprises segments of HIV-1. In an optional embodiment, the recombinant antigen further comprises segments of HIV-1 gp41.

[0015] In some embodiments of the present disclosure, the recombinant antigen further comprises a fusion partner.

[0016] Another aspect of the present disclosure provides a reagent / kit for detecting HIV, comprising the above-mentioned recombinant antigen.

[0017] Specifically, the recombinant antigen in the reagent / kit is coupled with a tracer marker or a solid phase carrier.

[0018] Yet another aspect of the present disclosure provides a nucleic acid molecule encoding the aforementioned HIV recombinant antigen, a vector containing the nucleic acid molecule, and a host cell containing the vector. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the expression vector map: the PE vector itself contains a 6×His tag at the N-terminus (other purification tags can also be used), so the translated HIV protein / recombinant protein all contains a 6-histidine tag, which can promote NI-NTA-assisted affinity purification. DETAILED DESCRIPTION

[0020] When used in conjunction with the term "comprising" in the claims and / or the specification, the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0021] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0022] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0023] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0024] Antigen reagents are used to detect sample antibodies. Antigen fragments used as reagents typically undergo certain technical processing. Therefore, the gp36 antigen described herein can be a full-length gp36 protein or a truncated gp36 protein. Exemplarily, the full-length gp36 protein comprises the amino acids shown in SEQ NO. 1, or a truncated gp36 protein based thereon, including amino acids X through Y of the gp36 protein, where X is selected from any integer between 1 and 60, and Y is selected from any integer between 143 and 161. Exemplary, but not limited to, amino acids 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, 60-161, etc. of gp36. For example, the gp36 antigen comprises a sequence that is 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% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0025] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments.

[0026] The term "fusion partner" is sometimes also referred to as a tag polypeptide, which is fused or connected to a protein of interest (e.g., HIV recombinant antigens of the present disclosure) and thereby promotes soluble expression, stabilization, and / or purification of the recombinant protein. The tag can be fused or connected to the N-terminus and / or C-terminus of the protein of interest (optionally via a linker or protease cleavage site). Such tags are well known to those skilled in the art and have been described in detail in the prior art literature. For example, such tags include, but are not limited to, histidine (His) tags, glutathione transferase (GST) tags, maltose binding protein (MBP) tags, thioredoxin (Trx) tags, NusA tags, disulfide isomerase DsbA tags, DsbC tags, SUMO tags, msyB tags, TF tags, initiator tags, ubiquitin tags, Myc tags, Flag tags, fluorescent proteins (e.g., GFP) tags, biotin tags, avidin tags, and Foldon domains.

[0027] The terms "peptide linker" and "connecting peptide" refer to short peptides used to connect two molecules (e.g., proteins). A connecting peptide may be present in the HIV chimeric antigens disclosed herein. Typically, a polynucleotide sequence encoding the short peptide is introduced (e.g., by PCR amplification or ligase) between two DNA fragments encoding the two target proteins to be connected, and protein expression is performed to obtain a fusion protein, such as target protein 1-connecting peptide-target protein 2, or more specifically, gp36 antigen-connecting peptide-fusion partner, or gp36 antigen-connecting peptide-other HIV fragment.

[0028] As used in this disclosure, the term "recombinant protein" refers to a protein obtained using recombinant DNA or recombinant RNA technology, which can be obtained in vivo or in vitro.

[0029] As used in this disclosure, the term "polypeptide" refers to any molecule comprising three or more amino acid residues linked by peptide bonds. Polypeptides according to the present application include peptides (e.g., tripeptides, oligopeptides, etc.), as well as peptides that may include chemical modifications such as saccharification (glycopolypeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation. Polypeptides may also refer to proteins.

[0030] As used herein, the term "signal peptide" refers to a polypeptide that is attached to a target protein and can promote the expression or transfer of the target protein. For example, the signal peptide can be attached to the N-terminus of the target protein and can usually be cleaved and removed, so that it is not present in the mature protein secreted by the cell.

[0031] As used in the present disclosure, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion or addition of one or more amino acids or nucleotides". In the present disclosure, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In some embodiments, the "mutation" of the present disclosure may be selected from "conservative mutation", "semi-conservative mutation" and "non-conservative mutation". In the present disclosure, the term "non-conservative mutation" or "semi-conservative mutation" may be a mutation that causes loss of function or partial loss of protein function. The term "conservative mutation" refers to a mutation that can maintain the function of a protein normally. A representative example of a conservative mutation is a conservative substitution.

[0032] As used in this disclosure, "conservative substitutions" generally exchange an amino acid at one or more sites in a protein. Such substitutions can be conservative. Specifically, substitutions considered to be conservative substitutions include the substitution of Ala to Ser or Thr, the substitution of Arg to Gln, His or Lys, the substitution of Asn to Glu, Gln, Lys, His or Asp, the substitution of Asp to Asn, Glu or Gln, the substitution of Cys to Ser or Ala, the substitution of Gln to Asn, Glu, Lys, His, Asp or Arg, the substitution of Glu to Gly, Asn, Gln, Lys or Asp, the substitution of Gly to Pro, the substitution of His to Asn, Lys, Gln, Arg or Tyr, the substitution of Ile to Leu, Substitution of Met, Val or Phe, substitution of Leu for Ile, Met, Val or Phe, substitution of Lys for Asn, Glu, Gln, His or Arg, substitution of Met for Ile, Leu, Val or Phe, substitution of Phe for Trp, Tyr, Met, Ile or Leu, substitution of Ser for Thr or Ala, substitution of Thr for Ser or Ala, substitution of Trp for Phe or Tyr, substitution of Tyr for His, Phe or Trp, and substitution of Val for Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations due to individual differences, strain differences, species differences, etc., from which the gene is derived.

[0033] "Sequence identity" and "percentage identity" in the present disclosure refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. A polynucleotide can differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). A polypeptide can differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). 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, percent 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.

[0034] Illustratively, in the present disclosure, two or more sequences or subsequences have "sequence identity" or "percent identity" of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide or amino acid residues when compared and aligned using a sequence comparison algorithm or as measured by visual inspection for maximum correspondence. The determination / calculation of "sequence identity" or "percent identity" can be based on any suitable region of the sequence. For example, a region of at least about 50 residues in length, a region of at least about 100 residues in length, a region of at least about 200 residues in length, a region of at least about 400 residues in length, or a region of at least about 500 residues in length. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (i.e., nucleic acids or polypeptides).

[0035] As used in the present disclosure, the term "polynucleotide" refers to a polymer consisting of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, derived from a nucleotide sequence separated at least once in quantity or concentration, capable of identifying, manipulating, and recovering sequences and their component nucleotide sequences by standard molecular biology methods (e.g., using a cloning vector). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), wherein "U" replaces "T." In other words, a "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (separate fragment or entire fragment), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. "Recombinant polynucleotides" and "recombinant nucleic acid molecules" belong to a type of "polynucleotide."

[0036] As used herein, the term "recombinant nucleic acid molecule" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform into a suitable host cell. The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide," "recombinant protein," "fusion protein," and the like.

[0037] As used in this disclosure, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to appropriate control sequences to express a gene of interest in a suitable host.

[0038] As used herein, the term "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcriptional and translational initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmids, viruses, bacteriophages, and transposons.

[0039] The term "host cell" in this disclosure refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a gene editing element, nucleic acid construct, or recombinant expression vector of the present disclosure. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the introduction of a gene editing element, nucleic acid construct, or recombinant expression vector, and recombinant host cells are specifically achieved by transformation. The host cell of the present disclosure can be a prokaryotic cell or a eukaryotic cell, as long as it is a cell that can be introduced with a recombinant nucleic acid molecule or recombinant expression vector of the present disclosure.

[0040] As used herein, the terms "transformation," "transfection," and "transduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0041] As used in this disclosure, the terms "sample," "specimen," "test sample," or "test sample" refer to any type of sample for which it is desired to determine whether it contains HIV antibodies. Exemplary, the test sample can be any product produced by a subject, or any product derived from a product produced by a subject. The sample can be taken from any tissue or body fluid, such as a blood sample (including a sample obtained from blood), a serum sample, a lymph sample, a saliva sample, or a synovial fluid. The sample obtained from blood can be a selected portion of the patient's blood or the vaccine recipient's blood, such as a selected cell-containing portion, or a plasma or serum portion. In some embodiments, the sample can be any sample containing antibody products of a humoral immune response.

[0042] As used in this disclosure, "diagnosis" includes the detection or identification of a disease state or condition in a subject, determining the likelihood that a subject will develop a given disease or condition, determining the likelihood that a subject with a disease or condition will respond to treatment, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and determining the effect of treatment on a subject with a disease or condition. For example, diagnostics can be used to detect the presence or likelihood of HIV infection in a subject or the likelihood that such a subject will respond favorably to a compound (e.g., a drug, e.g., pharmaceutical) or other treatment.

[0043] The terms "individual," "patient," or "subject" as used herein include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0044] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo, or the products can exist in an in vitro, ex vivo, or in vivo form. The term "in vitro" refers to experiments using materials, biological substances, cells and / or tissues under laboratory conditions or in culture medium; while the term "in vivo" refers to experiments and procedures using intact multicellular organisms. In some embodiments, the methods performed in vivo can be performed on non-human animals. "Ex vivo" refers to an event that exists or occurs outside an organism, such as outside a human or animal, such as an event that can exist or occur on a tissue (such as a whole organ) or cell taken from an organism.

[0045] Technical Solution

[0046] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 through substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 1.

[0047] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 2.

[0048] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 3.

[0049] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:4, or a polypeptide having an amino acid sequence as shown in SEQ ID NO:4 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO:4.

[0050] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 5, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 5 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 5.

[0051] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 6.

[0052] In some optional embodiments, the HIV recombinant antigen comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 7, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 7 by substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 7.

[0053] In some optional embodiments, the connecting peptide may be a flexible connecting peptide, including but not limited to GS, GGSGG (SEQ ID NO.8), KESGSVSSEQLAQFRSLD (SEQ ID NO.9), GSAGSAAGSGEF (SEQ ID NO.10), GGSGGEFGGSGG (SEQ ID NO.11), etc.

[0054] In some embodiments, the recombinant HIV antigen further comprises a tag polypeptide at its N-terminus and / or C-terminus. Exemplarily, the tag polypeptide is a His tag, preferably a 6×His tag, attached to the N-terminus of the HIV antigen, enabling purification of the recombinant HIV antigen using Ni-NTA affinity chromatography. In some alternative embodiments, the recombinant HIV antigen and the tag polypeptide may be linked by a linker peptide.

[0055] In some embodiments, the HIV recombinant antigen is coupled to a tracer marker or a solid phase carrier; the tracer marker is selected from at least one of acridinium ester, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, colloidal gold, fluorescent microspheres, terpyridine ruthenium, quantum dot luminescent materials and upconversion luminescent materials; the solid phase carrier is selected from at least one of magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

[0056] In some embodiments, the present disclosure provides a recombinant nucleic acid molecule comprising a nucleotide sequence encoding any of the above-mentioned HIV recombinant antigens. The recombinant nucleic acid molecule can be DNA, RNA, or a combination thereof.

[0057] In some embodiments, the present disclosure provides a recombinant expression vector comprising the aforementioned recombinant nucleic acid molecule, which can be used to transfer the recombinant nucleic acid molecule into a cell. In some embodiments, the recombinant expression vector comprises one or more regulatory elements, which can be promoters, enhancers, silencers, insulators, and other elements commonly used in the art. The regulatory elements are operably linked to the recombinant nucleic acid molecule to mediate transcription and translation of the recombinant nucleic acid molecule.

[0058] The term "operably linked" can include situations where a selected nucleic acid sequence is covalently linked to a regulatory element sequence (e.g., a promoter and / or enhancer) to place the expression of the nucleic acid sequence under the influence or control of the regulatory element (thereby forming an expression cassette). Thus, a regulatory element is operably linked to a nucleic acid molecule if the regulatory element is capable of effecting transcription of the nucleic acid molecule. The resulting transcript can be translated into a desired polypeptide or protein.

[0059] In the present 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 murine leukemia virus (MLV)), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), etc.

[0060] In some embodiments, the vector is a prokaryotic vector comprising various elements for expression in prokaryotic cells. For example, vectors include, but are not limited to, pET series vectors, Duet series vectors, pGEX series vectors, pHY300 vectors, pHY300PLK vectors, and PE vectors. Preferably, the vector is a PE vector.

[0061] In some embodiments, the present disclosure provides a recombinant host cell comprising the aforementioned recombinant nucleic acid molecule or recombinant expression vector. The recombinant host cell is obtained by transforming, transfecting, or transducing the recombinant nucleic acid molecule or recombinant expression vector into a host cell. The host cell in the present disclosure can be a eukaryotic cell or a prokaryotic cell, as long as it can be introduced with the recombinant nucleic acid molecule or recombinant expression vector of the present disclosure to achieve expression of the recombinant receptor binding protein.

[0062] In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. The bacterium can be a coccus (e.g., Micrococcus, Thermococcus, Streptococcus), a bacillus (e.g., Enterobacter, Bacillus, Actinomyces, Acetobacter, Lactobacillus), or a spirochete (e.g., Brachyspira). In some embodiments, the host cell is Escherichia coli ER2529, Escherichia coli BL21 (DE3), etc.; preferably, Escherichia coli ER2566.

[0063] In some embodiments, HIV recombinant antigens can be prepared by expressing in recombinant host cells, which can be prokaryotic or eukaryotic cells. In some embodiments, the recombinant host cell is a prokaryotic cell, for example, a recombinant host cell obtained by introducing a recombinant expression vector into Escherichia coli.

[0064] The recombinant host cells are cultured under conditions suitable for protein expression. After the culture is completed, the recombinant HIV antigen protein is collected from the cell culture fluid or fermentation fluid, and then the HIV recombinant antigen protein is purified.

[0065] In some embodiments, the step of protein purification of the HIV recombinant antigen protein includes Ni-NTA affinity chromatography, ion exchange chromatography, and protein dialysis.

[0066] In some embodiments, the present disclosure provides a method for diagnosing HIV-related diseases, comprising: contacting the aforementioned HIV recombinant antigen, reagent, or kit with HIV antibodies in a biological sample, and detecting the presence of HIV-1 antibodies in the biological sample.

[0067] In some optional embodiments, the above method comprises performing an immunoassay on the biological sample, wherein the immunoassay comprises ELISA, fluorescent immunochromatography, colloidal gold immunochromatography, chemiluminescence assay, electrochemiluminescence assay, indirect immunofluorescence assay (IFA) or radioimmunoassay (RIA).

[0068] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer are generally followed. Materials and reagents used in the examples were obtained through commercial channels unless otherwise specified.

[0069] Example 1 Preparation of HIV recombinant antigen

[0070] 1.1 Preparation of gp36 recombinant antigen

[0071] Plasmid design

[0072] According to the sequences shown in SEQ NO. 2, SEQ NO. 3, and SEQ NO. 4, respectively, the fusion partner GST was introduced at the N-terminus of the recombinant antigen, and expression plasmids were constructed based on the nucleic acid encoding the protein sequence. The gene sequence contains restriction enzyme sites BamHI and EcoRI at both ends. The mutant gene was treated with restriction endonucleases BamHI and EcoRI, respectively, and the treated gene fragment was ligated into a PE vector (as shown in Figure 1) to obtain the corresponding expression plasmid.

[0073] Inducible expression

[0074] The constructed expression plasmid was transformed into the Escherichia coli expression strain ER2566 (NEB) using the heat shock method. The plasmid was plated onto LB plates containing 100 μg / ml Amp and incubated at 37°C for 16 hours. Single colonies were selected, and positive strains identified by PCR were selected for sequencing. After sequencing, the strains were inoculated into LB medium containing 50 μg / ml Amp and incubated at 37°C with shaking. When the OD600 reached 0.6-0.8, 1.0 mM IPTG was added and the cells were induced at 37°C for 2-4 hours. The recombinant protein expression was analyzed by SDS-PAGE. After purification, the resulting proteins were named HIV-G-1 (SEQ NO. 2), HIV-G-2 (SEQ NO. 3), and HIV-G-3 (SEQ NO. 4).

[0075] 1.2 Preparation of gp36-gp41 Chimeric Antigen

[0076] A fusion expression antigen of gp36 (SEQ NO. 4) and gp41 (SEQ NO. 5) was prepared, i.e., a gp41-gp36 chimeric sequence (SEQ NO. 5 + SEQ NO. 4, with an enzyme cleavage site amino acid residue RS between the two) was designed, and the recombinant protein HIV-G-4 (SEQ NO. 6) was obtained in the same manner as in Example 1.1.

[0077] 1.3 Preparation of cysteine ​​mutant antigens

[0078] A cysteine ​​mutant antigen (wherein cysteine ​​was mutated to serine) was designed based on HIV-G-4, and the recombinant protein HIV-G-5 (SEQ NO. 7) was obtained in the same manner as in Example 1.1.

[0079] Example 2 Evaluation of the sensitivity and specificity of the disclosed recombinant HIV antigen using a double antigen sandwich method on a colloidal gold chromatography platform

[0080] 2.1 HIV-2 sample testing

[0081] (1) HIV antigen marker

[0082] Take 5ml of 4 / 10,000 colloidal gold, add an appropriate amount of 0.2M K2CO3, stir for 5 minutes, add HIV-labeled antigen, stir for 5 minutes, and then add an appropriate amount of 10% BSA to block and terminate labeling; centrifuge at 10,000 rpm for 10 minutes, remove the supernatant, re-dissolve the precipitate with gold reconstitution solution, and finally adjust the volume to 0.5ml (i.e., 1 / 10 the volume of the colloidal gold solution) with gold reconstitution solution; finally dilute the labeled antigen concentrated gold with the gold reconstitution solution by a certain multiple to prepare the gold working solution, and spread the gold; lyophilize the spread gold in a freeze dryer (1-2 hours) or dry it in a 37°C drying room overnight;

[0083] (2) HIV antigen coating

[0084] HIV antigen was diluted to 1.0 mg / ml with coating diluent (10 mm PB + 150 mm NaCl + 2% sucrose, pH 7.4) and coated on T-line and placed in a 37°C incubator for 1-2 h;

[0085] (3) Preparation of gold label:

[0086] Use a strip cutter to cut the gold label strips into the desired width, assemble them, and then add the sample for testing. It should be noted that the colloidal gold card has 10 reading gradients: C1, C2, C3, C4, C5, C6, C7, C8, C9, and B. These 10 gradients correspond to decreasing T-line color from left to right: C1 represents the darkest T-line color, C9 represents the lightest T-line color, B represents no color, and B+ represents almost no color. In Table 2, "C3+" indicates a color slightly darker than C3 but not reaching C2, and the same applies to other values ​​marked with "+."

[0087] Test results:

[0088] The color rendering degree is judged by the color card, and the results are as follows:

[0089] The results demonstrated that HIV-G-1, HIV-G-2 and HIV-G-3 disclosed herein as antigens can detect HIV-2 samples with high sensitivity and specificity.

[0090] 2.2 HIV sample testing (including confirmed HIV-2 positive samples and Abbott HIV positive samples)

[0091] The operation process is the same as that of Example 2.1, except that two T lines are coated on the test strip with P81 antigen against HIV-1 and P82 antigen against HIV-2, respectively.

[0092] Test results:

[0093] The color development degree is judged by the color card, and the results are as follows (the reading on the left of the symbol " / " indicates the test result of HIV-2 antibodies, and the reading on the right indicates the test result of HIV-1 antibodies):

[0094] The results showed that HIV-G-4 and HIV-G-5 disclosed in the present invention can be used as antigens to detect HIV-positive samples well.

[0095] Example 3 Thermal Stability Investigation

[0096] The stability assessment of HIV recombinant antigens included accelerated testing at 37°C for 3 days and 7 days, and detection of aggregate formation by SDS-PAGE protein gel without mercaptoethanol treatment.

[0097] 3.1 Aggregate formation

[0098] The following table summarizes the aggregates observed by SDS-PAGE of HIV recombinant antigens stored at 4°C and then at 37°C for 3 and 7 days without mercaptoethanol treatment. Fewer aggregates indicate better stability. This indicates that the cysteine ​​to serine mutation in the HIV recombinant antigen significantly improves thermal stability.

[0099] Related sequences

[0100] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0101] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure. Industrial Applicability

[0102] The inventors analyzed the gp36 proteins of different HIV-2 strains and designed the gp36 protein sequence shown in SEQ NO. 1. The recombinant HIV antigen designed based on this sequence can effectively detect clinical HIV-2 positive samples with high sensitivity and specificity. Using this recombinant HIV antigen protein as a labeling end significantly enhances the marker activity of the recombinant HIV antigen, reduces nonspecific immune binding activity, and improves sensitivity.

Claims

1. A HIV recombinant antigen, wherein: The HIV recombinant antigen comprises a segment of amino acids from position x to position y of HIV-2 gp36 protein, wherein x is any integer from 1 to 60, and y is any integer from 143 to 161, wherein the amino acid sequence of the HIV-2 gp36 protein is shown in SEQ NO.

1.

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

4.

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

5. The recombinant antigen according to claim 1, wherein The recombinant antigen also contains segments of other HIV proteins. Optionally, the recombinant antigen also contains segments of HIV-1. Optionally, the recombinant antigen also contains segments of HIV-1gp41. Optionally, the recombinant antigen contains the HIV-1gp41 protein sequence as shown in SEQ NO.

5. Optionally, the recombinant antigen also contains 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 NO.6 or SEQ NO.

7.

7. A HIV recombinant antigen, wherein: The HIV recombinant antigen comprises any of the following polypeptides: a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 through substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 1; b) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2 which has been substituted, repeated, deleted or added with one or more amino acids and has or partially has the activity of the polypeptide having the sequence as shown in SEQ ID NO: 2; c) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 3 which has been substituted, repeated, deleted or added with one or more amino acids, and has or partially has the activity of the polypeptide having the sequence as shown in SEQ ID NO: 3; d) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 4, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 4 with one or more amino acids substituted, repeated, deleted or added, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 4; e) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 5, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 5 through substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 5; f) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6, or a polypeptide having an amino acid sequence as shown in SEQ ID NO: 6 through substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO: 6; g) a polypeptide having an amino acid sequence as shown in SEQ ID NO:7, or a polypeptide having an amino acid sequence as shown in SEQ ID NO:7 through substitution, duplication, deletion or addition of one or more amino acids, and having or partially having the activity of the polypeptide having the sequence as shown in SEQ ID NO:

7.

8. A reagent for detecting HIV, wherein: The reagent comprises the recombinant antigen according to any one of claims 1 to 7.

9. The reagent according to claim 8, wherein The recombinant antigen is coupled with a tracer marker or a solid phase carrier; the tracer marker is selected from at least one of acridinium ester, luminol, isoluminol, alkaline phosphatase, horseradish peroxidase, colloidal gold, fluorescent microspheres, terpyridine ruthenium, quantum dot luminescent materials and upconversion luminescent materials; the solid phase carrier is selected from at least one of magnetic microspheres, plastic microspheres, plastic particles, microplates, glass, capillaries, nylon and nitrocellulose membranes.

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

11. A nucleic acid molecule encoding the 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 the recombinant antigen according to any one of claims 1 to 7 or the reagent according to any one of claims 8 to 9 in preparing a kit for detecting HIV.

13. A method for diagnosing HIV-related diseases, comprising: Contacting the HIV recombinant antigen according to any one of claims 1 to 7, the reagent according to any one of claims 8 to 9, or the kit according to claim 10 with HIV antibodies in a biological sample to detect the presence of HIV-1 antibodies in the biological sample; Optionally, the method comprises performing an immunoassay on the biological sample, wherein the immunoassay comprises ELISA, fluorescent immunochromatography, colloidal gold immunochromatography, chemiluminescence assay, electrochemiluminescence assay, indirect immunofluorescence assay (IFA) or radioimmunoassay (RIA).

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