Recombinant antigens for detecting HIV antibodies

Abstract

The invention provides an HIV recombinant antigen for detecting an HIV antibody, and belongs to the technical field of immunodiagnosis. The invention particularly relates to an HIV (Human Immunodeficiency Virus) recombinant antigen, a nucleic acid molecule, an expression vector, a host cell, a reagent, a kit and application. According to the invention, the recombinant HIV antigen protein is used for a labeled terminal, so that the marker activity of the recombinant HIV antigen can be remarkably improved, the non-specific immune binding activity can be reduced, and the sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunodiagnosis and relates to a recombinant antigen for detecting HIV antibodies. Background Art

[0002] There are currently two types of HIV (human immunodeficiency virus), namely HIV-1 and HIV-2. HIV-1 is currently the main virus that causes ADIS. According to 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. According to different epidemic areas, M type can be divided into subtype A, B, C, D, E, F, G, H, I, J, K, and L. The transmission ability and toxicity of HIV-2 are weaker than those of HIV-1. There are currently 8 known types of HIV-2, and only 2 types have caused large-scale epidemics, namely group A and group B.

[0003] HIV virus is a spherical particle with a diameter of 100 to 120 nm, consisting of a core and an envelope. Its inner region consists of a conical core and is wrapped by the structural protein P24 (capsid). The core consists of the ssRNA genome (wrapped by the p7nucleocapsid), reverse transcriptase, integrase and protease, some minor proteins and the main core protein. A limited number of HIV envelope glycoproteins (Env) can be found on the surface of the virion, which are responsible for binding to its main host receptor CD4 and its co-receptors (mainly CCR5 or CXCR4), causing the virus to enter its target cells. The env gene of HIV-2 encodes the gp160 precursor protein, which is cleaved into gp125 protein and gp36 protein by the host cell protease furin in the host cell.

[0004] HIV antibody detection is one of the main methods for screening HIV-infected patients, and gp36 protein is the main antigen raw material for HIV-2 antibody detection. However, HIV gp36 is very hydrophobic, and it is usually difficult to prepare products with good detection performance. Therefore, designing a reasonable gp36 segment and optimizing the protein physicochemical properties of gp36 itself have important guiding significance for gp36 in vitro expression, storage and detection applications. Summary of the invention

[0005] In order to solve the problem that it is difficult to develop high-quality diagnostic products based on gp36 protein, the inventors analyzed the gp36 proteins of different HIV-2 strains and creatively designed the gp36 protein sequence shown in SEQ NO.1.

[0006] In one aspect of the present invention, a gp36 protein sequence as shown in SEQ NO. 1 for detecting HIV-2 antibodies is provided.

[0007] In some embodiments of the present invention, 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.

[0008] 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.

[0009] In some embodiments of the present invention, 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.

[0010] In some embodiments of the present invention, the cysteine ​​in the recombinant antigen is mutated to other amino acids, preferably, the cysteine ​​is mutated to serine.

[0011] In some embodiments of the present invention, the recombinant antigen further comprises segments of other HIV proteins. Preferably, the recombinant antigen further comprises segments of HIV-1. Preferably, the recombinant antigen further comprises segments of HIV-1 gp41.

[0012] In some embodiments of the present invention, the recombinant antigen further comprises a fusion partner.

[0013] Another aspect of the present invention provides a reagent / kit for detecting HIV, comprising the recombinant antigen according to any one of claims 1 to 6.

[0014] Specifically, the recombinant antigen in the reagent / kit is coupled with a tracer marker or a solid phase carrier.

[0015] Another aspect of the present invention 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.

[0016] Effects of the Invention

[0017] The HIV recombinant antigen designed by the present invention can effectively detect clinical HIV-2 positive samples with high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The expression vector map is shown: 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

[0019] 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".

[0020] 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.

[0021] 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.

[0022] 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 the alternatives are mutually exclusive.

[0023] When using antigen reagents to detect sample antibodies, antigen fragments used as reagents are usually processed by some techniques, so the gp36 antigen described in the present disclosure can be a full-length gp36 protein or a truncated gp36 protein. Exemplarily, the full-length gp36 protein contains amino acids as shown in SEQ NO.1, or a truncated gp36 protein based thereon, including amino acids from position X to position Y of the gp36 protein, wherein X is selected from any integer from 1 to 60, and Y is selected from any integer from 143 to 161. Exemplary, non-limiting, include, for example, 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.

[0024] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen binding site, covering 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, complete antibodies, and antibody fragments.

[0025] The term "fusion partner" is sometimes also referred to as a tag polypeptide, which is fused or connected to a target protein (e.g., HIV recombinant antigen of the present disclosure), and thereby promotes soluble expression, stabilization, and / or purification of the recombinant protein. The tag may be fused or connected 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 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.

[0026] The terms "peptide linker" and "connecting peptide" refer to short peptides used to connect two molecules (such as proteins). A connecting peptide may be present in the HIV chimeric antigen of the present invention. Generally, a fusion protein is obtained by introducing (for example, by PCR amplification or ligase) a polynucleotide sequence encoding the short peptide between two DNA fragments encoding two target proteins to be connected, respectively, and performing protein expression, such as target protein 1-connecting peptide-target protein 2, more specifically, gp36 antigen-connecting peptide-fusion partner, gp36 antigen-connecting peptide-other HIV fragments.

[0027] As used in the present 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.

[0028] As used in the present 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.), and peptides that may include chemical modifications (e.g., glycosylation (glycopolypeptides), phosphorylation, hydroxylation, sulfonation, palmitoylation, and disulfide bond formation). Polypeptide may also refer to protein.

[0029] As used in the present disclosure, the term "signal peptide" refers to a polypeptide that is connected to a target protein and can promote the expression or transfer of the target protein. Exemplarily, the signal peptide can be connected to the N-terminus of the target protein and can usually be cut and removed, so it is not present in the mature protein secreted by the cell.

[0030] 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 a nucleotide sequence or an 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 normally maintain the function of a protein. A representative example of a conservative mutation is a conservative substitution.

[0031] As used in the present disclosure, "conservative substitutions" generally exchange an amino acid at one or more sites in a protein. Such substitutions may be conservative. Specifically, substitutions considered to be conservative substitutions include 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. Conservative mutations include substitutions of Ile to Leu, Met, Val or Phe, substitutions of Leu to Ile, Met, Val or Phe, substitutions of Lys to Asn, Glu, Gln, His or Arg, substitutions of Met to Ile, Leu, Val or Phe, substitutions of Phe to Trp, Tyr, Met, Ile or Leu, substitutions of Ser to Thr or Ala, substitutions of Thr to Ser or Ala, substitutions of Trp to Phe or Tyr, substitutions of Tyr to His, Phe or Trp, and substitutions of Val to Met, Ile or Leu. In addition, conservative mutations also include naturally occurring mutations caused by individual differences, strains, species differences, etc. from which genes are derived.

[0032] "Sequence identity" and "percentage of 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. The polynucleotides may 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). The polypeptides may 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, 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 nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0033] Exemplary, in the present disclosure, 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% "sequence identity" or "percent identity" of nucleotides or amino acid residues when compared and aligned using a sequence comparison algorithm or measured by visual inspection with 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, a region of at least about 200 residues, a region of at least about 400 residues, or a region of at least about 500 residues. In certain embodiments, the sequences are substantially identical over the entire length of any one or two compared biopolymers (i.e., nucleic acids or polypeptides).

[0034] As used in the present disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and recovered by standard molecular biological methods (e.g., using a cloning vector) and sequences and their component nucleotide sequences. 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 fragments or entire fragments), 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 one of "polynucleotides".

[0035] As used in the present disclosure, 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", etc.

[0036] As used in this disclosure, the term "vector" refers to a DNA construct that contains a DNA sequence operably linked to appropriate control sequences so as to express a gene of interest in a suitable host.

[0037] As used in this disclosure, the term "recombinant expression vector" refers to a DNA structure used to express a polynucleotide encoding a desired polypeptide, for example. 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 transcription and translation start and stop sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, bacteriophages, and transposons.

[0038] The term "host cell" in the present disclosure means any cell type that is easily transformed, transfected, transduced, etc. with a gene editing element, nucleic acid construct or recombinant expression vector comprising the present disclosure. The term "recombinant host cell" covers 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 the recombinant host cell is 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 introduce a recombinant nucleic acid molecule or a recombinant expression vector of the present disclosure.

[0039] The terms "transformation, transfection, and transduction" in this disclosure 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 of introducing nucleic acid into a cell, including but not limited to 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.

[0040] As used in the present disclosure, the terms "sample", "specimen", "sample to be tested" or "test sample" refer to any type of sample for which it is desired to determine whether it contains HIV antibodies. Exemplary, the sample to be tested 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.

[0041] 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 a treatment, determining the prognosis of a subject with a disease or condition (or its likely progression or regression), and determining the effect of a treatment on a subject with a disease or condition. For example, a diagnosis can be used to detect the presence or likelihood of a subject being infected with HIV or the likelihood that such a subject will respond favorably to a compound (e.g., a drug, such as a pharmaceutical) or other treatment.

[0042] The terms "individual", "patient" or "subject" used in the context of this disclosure 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).

[0043] 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 events that exist or occur outside an organism, such as outside a human or animal, such as events that can exist or occur on tissues (e.g., whole organs) or cells taken from an organism.

[0044] Technical Solution

[0045] In some preferred 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.

[0046] In some preferred 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.

[0047] In some preferred 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.

[0048] In some preferred 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 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:4.

[0049] In some preferred 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 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.

[0050] In some preferred 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 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.

[0051] In some preferred 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 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.

[0052] In some preferred embodiments, the connecting peptide can be selected as a flexible connecting peptide, including but not limited to GS, GGSGG, KESGSVSSEQLAQFRSLD, GSAGSAAGSGEF, GGSGGEFGGSGG, etc.

[0053] In some embodiments, the HIV recombinant 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, which is connected to the N-terminus of the HIV antigen, so that the HIV recombinant antigen can be purified by NI-NTA affinity chromatography. In some optional embodiments, the HIV recombinant antigen and the tag polypeptide can be connected by a connecting peptide.

[0054] 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.

[0055] 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, and the recombinant nucleic acid molecule can be DNA, RNA or a combination thereof.

[0056] In some embodiments, the present disclosure provides a recombinant expression vector comprising the above-mentioned 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 element is operably connected to the recombinant nucleic acid molecule to mediate the transcription and translation of the recombinant nucleic acid molecule.

[0057] The term "operably linked" can include a situation where a selected nucleic acid molecule 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, if the regulatory element is capable of acting on the transcription of the nucleic acid molecule, the regulatory element is operably linked to the nucleic acid molecule. The resulting transcript can be translated into a desired polypeptide or protein.

[0058] 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, adenovirus-associated viral vectors, vaccinia virus vectors and herpes virus vectors), transposon vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), etc.

[0059] In some embodiments, the vector is a prokaryotic vector, which contains 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. Preferably, the vector is a PE vector.

[0060] In some embodiments, the present disclosure provides a recombinant host cell, which comprises the above-mentioned recombinant nucleic acid molecule or recombinant expression vector. The recombinant host cell is obtained by transforming, transfecting, or transducing a recombinant nucleic acid molecule or a recombinant expression vector into a host cell. The host cell in the present disclosure may be a eukaryotic cell or a prokaryotic cell, as long as the recombinant nucleic acid molecule or recombinant expression vector of the present disclosure can be introduced to achieve the expression of the recombinant receptor binding protein.

[0061] 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, Actinobacillus, 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.

[0062] In some embodiments, HIV recombinant antigens can be prepared by expressing in recombinant host cells, which can be prokaryotic cells or eukaryotic cells. In some embodiments, the recombinant host cells are prokaryotic cells, for example, the recombinant host cells are obtained by introducing a recombinant expression vector into Escherichia coli.

[0063] 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.

[0064] In some embodiments, the step of purifying the HIV recombinant antigen protein includes Ni-NTA affinity chromatography, ion exchange chromatography and protein dialysis.

[0065] The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0066] Example 1 Preparation of HIV recombinant antigen

[0067] 1.1 Preparation of gp36 recombinant antigen

[0068] Plasmid design

[0069] According to the sequences shown in SEQ NO.2, SEQ NO.3 and SEQ NO.4, the fusion partner GST was introduced at the N-terminus of the recombinant antigen, and the expression plasmid was constructed according to the coding nucleic acid of the protein sequence. The gene sequence contained 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 fragments were connected to the PE vector (such as Figure 1 To obtain the corresponding expression plasmid.

[0070] Inducible expression

[0071] The constructed expression plasmid was transformed into the E. coli expression strain ER2566 (NEB) by heat shock method, and coated on LB plate containing 100ug / ml Amp, and cultured at 37°C for 16h. Single colonies were picked, and positive strains identified by bacterial liquid PCR were selected for sequencing. After sequencing was correct, the strains were inoculated into LB medium containing 50ug / ml Amp and cultured at 37 degrees with shaking. After OD600 reached 0.6-0.8, 1.0mM IPTG was added, and the culture was induced at 37°C for 2-4h. The expression of recombinant proteins was identified by SDS-PAGE. After purification, the obtained proteins were named HIV-G-1 (SEQ NO.2), HIV-G-2 (SEQ NO.3) and HIV-G-3 (SEQ NO.4).

[0072] 1.2 Preparation of gp36-gp41 chimeric antigen

[0073] Prepare the fusion expression antigen of gp36 (SEQ NO.4) and gp41 (SEQ NO.5), that is, design the gp41-gp36 chimeric sequence (SEQ NO.5+SEQ NO.4, with an enzyme cleavage site amino acid residue RS between the two), and obtain the recombinant protein HIV-G-4 (SEQ NO.6) in the same manner as Example 1.1.

[0074] 1.3 Preparation of cysteine ​​mutant antigens

[0075] 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.

[0076] Example 2 Evaluation of the sensitivity and specificity of the recombinant HIV antigen of the present invention using the double antigen sandwich method on a colloidal gold chromatography platform

[0077] 2.1 HIV-2 sample testing

[0078] (1) HIV antigen marker

[0079] Take 5ml of 4 / 10,000 colloidal gold, add an appropriate amount of 0.2M K2CO3, stir for 5min, add HIV labeled antigen, stir for 5min, and then add an appropriate amount of 10% BSA to block and terminate the labeling; centrifuge at 10000rpm for 10min, remove the supernatant, re-dissolve the precipitate with gold re-solution, and finally use gold re-solution to make the volume to 0.5ml (i.e. 1 / 10 of the volume of colloidal gold solution); finally dilute the labeled antigen concentrated gold with a certain multiple with gold re-solution to prepare gold working solution, and spread the gold; put the spread gold into a freeze dryer for freeze drying (1-2h) or dry it in a 37℃ drying room overnight;

[0080] (2) HIV antigen coating

[0081] Dilute HIV antigen to 1.0 mg / ml with coating diluent (10 mm PB + 150 mm Nacl + 2% sucrose, pH 7.4) and coat the T line, and place in a 37°C incubator for 1 to 2 hours;

[0082] (3) Preparation of gold label:

[0083] Use a strip cutter to cut the gold label strip into strips of the required width, assemble it, and add the sample for testing. It should be noted that the readings of the colloidal gold card have 10 gradients, namely C1, C2, C3, C4, C5, C6, C7, C8, C9, and B, and the T line colors corresponding to these 10 gradients are lighter from left to right. C1 means the T line color is the darkest, C9 means the T line color is the lightest, B means no color, and B+ means almost no color; "C3+" in Table 2 means that the color is slightly darker than C3 but not as dark as C2, and the others with "+" are analogous.

[0084] Test results:

[0085] The color rendering degree is judged by the color card, and the results are as follows:

[0086]

[0087] The results show that HIV-G-1, HIV-G-2 and HIV-G-3 of the present invention can detect HIV-2 samples with high sensitivity and specificity as antigens.

[0088] 2.2 HIV sample testing (including confirmed HIV-2 positive samples and Abbott HIV positive samples)

[0089] 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.

[0090] Test results:

[0091] 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):

[0092]

[0093]

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

[0095] Example 3 Thermal Stability Study

[0096] The stability evaluation 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] By observing the HIV recombinant antigen stored at 4°C and tested at 37°C for 3 days and 7 days on SDS-PAGE protein gel without mercaptoethanol treatment, the aggregate situation is summarized in the following table. The fewer aggregates, the better the stability. It can be seen that the mutation of cysteine ​​in HIV recombinant antigen to serine can significantly improve thermal stability.

[0099]

[0100]

[0101] Related sequences

[0102] SEQ ID NO:1

[0103] LATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY

[0104] SEQ ID NO:2

[0105] LATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVF

[0106] SEQ ID NO:3

[0107] LSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVF

[0108] SEQ ID NO:4

[0109] AIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQ LEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY

[0110] SEQ ID NO:5

[0111] TLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIEESQNQQEKNEKDLLALDSWKNLWNWFDIT

[0112] SEQ ID NO:6

[0113] AIEKYLKDQAQLNSWGCAFRQVCHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY RSTLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIEESQNQQEKNEKDLLALDSWKNLWNWFDIT

[0114] SEQ ID NO:7

[0115] AIEKYLKDQAQLNSWGSAFRQVSHTTVPWVNDSLSPDWKNMTWQEWEKQVRYLEANISQQLEQAQIQQEKNMYELQKLNSWDVFSNWFDLTSWIKYIQYGVY RS TLTVQARQLLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLQDQQLLGIWGSSGKLISTTAVPWNSSWSNKSQDEIWDNMTWMQWDKEISNYTYEIYTLIEESQNQQEKNEKDLLALDSWKNLWNWFDIT。

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, and 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, preferably, the cysteine ​​is mutated to serine.

5. The recombinant antigen according to claim 1, wherein The recombinant antigen further comprises segments of other HIV proteins, preferably, the recombinant antigen further comprises segments of HIV-1, preferably, the recombinant antigen further comprises segments of HIV-1 gp41, preferably, the recombinant antigen further comprises a fusion partner.

6. A reagent for detecting HIV, wherein: The reagent comprises the recombinant antigen according to any one of claims 1 to 5.

7. The reagent according to claim 6, 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.

8. A kit for detecting HIV, comprising the recombinant antigen according to any one of claims 1 to 5, or the reagent according to any one of claims 6 to 7.

9. A nucleic acid molecule encoding the HIV recombinant antigen according to any one of claims 1 to 5, a vector containing the nucleic acid molecule, and a host cell containing the vector.

10. Use of the recombinant antigen according to any one of claims 1 to 5 or the reagent according to any one of claims 6 to 7 in preparing a kit for detecting HIV.

Images