HIV proteins and their use in detecting HIV antibodies

Abstract

The present invention provides a HIV protein that can be used as a more superior detection material for detecting the presence of HIV antibodies in a sample from a subject.

Description

Technical Field

[0001] This invention relates to the field of HIV antibody detection. Specifically, it relates to recombinant HIV proteins that can be used to detect the presence of HIV antibodies in samples from subjects. The invention also relates to nucleic acids encoding the aforementioned recombinant HIV proteins, as well as related vectors, host cells, immunoassay methods, and detection kits. Background Technology

[0002] Acquired immunodeficiency syndrome (AIDS) is a disease caused by infection with the human immunodeficiency virus (HIV). HIV targets the human immune system, and without any treatment, the immune system of an HIV-infected person will gradually be destroyed by HIV until they lose almost all of their immune capacity. The main modes of transmission for AIDS include blood transmission, mother-to-child transmission, and sexual transmission.

[0003] According to the World Health Organization, as of 2021, approximately 38.4 million people worldwide were living with HIV. In 2021, there were approximately 1.5 million new infections and 650,000 deaths. Of these, approximately 20.6 million people living with HIV lived in East and Southern Africa. As of 2020, approximately 36.3 million people worldwide had died from AIDS.

[0004] Currently, there are two types of HIV: HIV-1 and HIV-2. HIV-1 is the primary cause of HIV-1 infections. Based on different cross-species transmission chains, HIV-1 can be classified into: M, N, O, and P types. M type accounts for 90% of HIV-1 infections. Based on different epidemic regions, M type can be further divided into subtypes A, B, C, D, E, F, G, H, I, J, K, and L. HIV-2 has a lower transmissibility and less virulence than HIV-1. Currently, there are eight known HIV-2 subtypes, but only two types cause large-scale epidemics: group A and group B.

[0005] HIV is a spherical particle with a diameter of 100–120 nm, consisting of a core and an envelope. Its inner region comprises a cone-shaped core encapsulated by the structural protein P24 (capsid). The core consists of the ssRNA genome (encapsided by p7 nucleocapsid), enzymes such as reverse transcriptase, integrase, and protease, along with 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, responsible for binding to its main host receptor CD4 and its co-receptors (primarily CCR5 or CXCR4), leading to viral entry into its target cells. The HIV env gene encodes the gp160 precursor protein, which is cleaved by the host cell protease furin into gp120 and gp41, forming the active gp120-gp41 complex protein.

[0006] HIV antibody testing is one of the main methods for HIV infection screening. HIV protein is an important raw material for HIV antibody testing, which is related to the sensitivity and activity of the test. However, due to the difficulty in expressing the hydrophobic regions of HIV protein, the design and optimization of HIV protein is of great significance for providing better detection raw materials. Summary of the Invention

[0007] This invention provides a protein for detecting the presence of HIV antibodies in samples from subjects. The inventors designed and optimized the HIV protein to improve at least one of the following problems: low antibody detection activity or insufficient specificity, and difficulty in protein expression.

[0008] In some embodiments, the present invention provides a protein having at least one of the following mutations at the corresponding positions of the amino acid sequence shown in SEQ ID NO:1:

[0009] L30 mutates to M;

[0010] S83 mutates to N or V;

[0011] E86 mutates to D or Q;

[0012] E113 mutates to K or H;

[0013] N136 mutation to A or S.

[0014] In some embodiments, the protein has at least one of the following combinations of mutations at the corresponding positions of the amino acid sequence shown in SEQ ID NO:1:

[0015] Combination 1: L30M, S83V, E86D, E113H, N136A;

[0016] Combination 2: L30M, S83V, E86D, E113K, N136S;

[0017] Combination 3: L30M, S83N, E86Q, E113K, N136S;

[0018] Combination 4: L30M, S83N, E86D, E113H, N136S;

[0019] Combination 5: L30M, S83V, E86Q, E113H, N136A;

[0020] Combination 6: L30M, S83N, E86Q, E113K, N136A;

[0021] Combination 7: L30M, S83V, E86Q, E113K, N136S;

[0022] Combination 8: L30M, S83N, E86D, E113H, N136A.

[0023] In some implementation schemes, HIV proteins are prone to aggregate formation during in vitro expression due to their amino acid composition and hydrophobic properties, which can lead to problems such as short shelf life or changes in activity caused by thermal degradation. Introducing mutations can improve the stability of HIV proteins.

[0024] In some embodiments, the protein has one of the following mutations at the corresponding position of the amino acid sequence shown in SEQ ID NO:1: C63S or C69S.

[0025] The amino acid sequence of SEQ ID NO:1 is shown below:

[0026]

[0027] HIV-1 is the most prevalent type of virus, dominating in many parts of the world. In some embodiments, the HIV protein is used to bind to or detect HIV-1 antibodies. This is particularly suitable for the detection of HIV-1 infection in animals, especially mammals, particularly primates, and especially humans. In some embodiments, the protein is the HIV-1 gp41 antigenic peptide. In some embodiments, the protein also includes other different, independently defined HIV-1 peptides, such as the HIV-1 gp120 antigenic peptide.

[0028] In some embodiments, the N-terminus and / or C-terminus of the protein also have a fusion chaperone.

[0029] In some embodiments, the protein also has linkers at its N-terminus and / or C-terminus.

[0030] HIV-2 is prevalent in a few areas, and further testing is needed to detect the presence of HIV-2 antibodies. In some embodiments, the N-terminus and / or C-terminus of the protein also contain an HIV-2 antigenic peptide, such as a fragment of at least 25, 30, 40, 50, 70, 90, 110, 150, 200, 300, 400, or 500 amino acids from the Env region of HIV-2 virus (e.g., gp36, gp140); for example, all or part of HIV-2 gp36, such as positions 587-614, 538-662, 580-607, 521-661, and 580-607 of HIV-2 gp160, where positions XX-XX represent the position of the antigenic peptide on HIV-2 gp160. The relative positions may vary slightly between different HIV strains, approximately 10 amino acids. The gp36 antigenic peptide is, for example, AIEKYLKDQAQLNSWGCAFRQVCHTSVP (SEQ ID). NO:4), in some respects, cysteine ​​in gp36 is mutated to serine.

[0031] In some embodiments, the present invention also provides an amino acid sequence having at least 80%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with the protein.

[0032] In some embodiments, the present invention also provides a conjugate comprising the protein and the conjugated moiety. In some embodiments, the conjugated moiety is selected from solid phases, markers, or conjugates.

[0033] In some embodiments, the solid phase is selected from microspheres, plates, and membranes; in other embodiments, the solid phase is selected from magnetic microspheres, plastic microspheres, latex microparticles, microporous plates, glass, capillaries, nylon, or nitrocellulose membranes.

[0034] In some embodiments, the label is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense materials, chemiluminescent labels, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes. In some embodiments, the label is selected from fluorescent microspheres, colored latex microspheres, acridinium esters, alkaline phosphatase, horseradish peroxidase, or colloidal gold.

[0035] In some implementation plans, the Overseas Chinese Federation selects inert proteins, biotin, or avidin.

[0036] In some embodiments, the present invention provides nucleic acids for encoding the protein.

[0037] In some embodiments, the present invention provides a vector containing the nucleic acid.

[0038] In some embodiments, the present invention provides a host cell containing the vector.

[0039] In some embodiments, the present invention provides a kit comprising the protein or conjugate.

[0040] In some embodiments, the kit further includes other components that are mixed with the protein or conjugate. In some embodiments, such as a preservation solution, the protein or conjugate is dispersed in the preservation solution. In some embodiments, the preservation solution includes at least one suitable buffering agent, salt, sugar, other unrelated proteins (e.g., non-HIV proteins), surfactant, etc.

[0041] In some embodiments, the kit also includes additional HIV proteins. It is used in some aspects for the detection of HIV antibodies in samples using a double-antigen sandwich assay.

[0042] In some embodiments, the kit further includes a secondary antibody. In some aspects, it is used for indirect or capture methods to detect HIV antibodies in a sample. In some aspects, the secondary antibody is an anti-human antibody.

[0043] In some embodiments, the protein or conjugate is used in the preparation of a kit or a kit for detecting HIV antibodies.

[0044] In some embodiments, the present invention provides a detection method comprising contacting a protein or conjugate or a kit with a sample. In some embodiments, the detection method is used to detect HIV antibodies in a sample.

[0045] In some embodiments, the sample includes biological tissues, cells, or body fluids in a healthy or pathological state, such as blood samples, such as plasma, serum, blood products, such as semen, or vaginal secretions.

[0046] The term "amino acids" in this article includes both naturally occurring and non-naturally occurring amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0047] Regarding the description of mutations, for example, L30M in this article refers to the mutation of the 30th amino acid in the sequence shown from leucine (leu, L) to methionine (met, M). Other similar mutation descriptions are understood in the same way.

[0048] The term "fusion partner" in this article can generally refer to any protein or small molecule. Any fusion partner of interest can be used, which may have functions such as improving purification, enhancing expression, facilitating coupling, and stabilizing peptides. Fusion partners can be carrier proteins (e.g., BSA), histidine tags, biotin tags; or some solubilizing tags such as mannose-binding protein (MBP), thioreductase (Trx), NusA, glutathione S-transferase (GST), etc.

[0049] The term "linker" is used in this context to refer to the sequential linking of two or more proteins from the N-terminus to the C-terminus, and is often also called a linker. Suitable peptide linkers can have a length of about 1 to about 50 amino acids, for example, about 1 to about 30 amino acids, or about 4 to 20 amino acids. Examples include (G)n, (GS)n, (SG)n, (GGGS)n, (G4S)n, (SG4)n, and G4(SG4)n, where n is typically a value between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, usually between 1 and 4; and also such as GGGSG, GGSGG, GSGGG, or SGGGG. Other examples may include peptides containing amino acid residues other than G or S, such as GGTGS, GTSPGG, GNGGGS, etc.

[0050] The terms "protein" and "peptide" are used interchangeably in this document. Proteins or peptides can be natural or non-natural. Proteins and peptides are not required to have tertiary or quaternary structures. Proteins or peptides can be obtained synthetically or through an expression system. An expression system can be a prokaryotic host cell system, such as *Escherichia coli*, which has been modified to express the proteins or peptides of this invention. In alternative embodiments, the expression system is a eukaryotic host cell system, such as yeast, or mammalian cells, such as CHO cells or HEK 293 cells. Suitable expression systems are known in the art and can be prepared or commercially obtained using known techniques. Proteins or peptides can be natural or non-natural.

[0051] Sequence “identity” or “homology” is defined in this document as the percentage of amino acid residues in a candidate sequence that are identical to those in a specific protein or peptide sequence after aligning a candidate sequence with a specific protein or peptide sequence (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved mutations as part of the sequence identity. Various methods in the art can be used to determine the percentage of amino acid sequence identity, such as publicly available computer software like BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0052] The term "conjugate" in this document refers to any substance formed by joining independent parts together, and can be conjugated in any manner. Representative conjugates of this invention include those formed by joining HIV proteins with conjugate components such as solid phases, markers, and conjugates. In conjugates, conjugation can occur through one or more active sites, and can occur directly or indirectly. The "solid phase" can be made of any material, including microspheres, plates, and membranes; for example, magnetic microspheres, plastic microspheres, latex microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes. The "marker" refers to a substance capable of being directly or indirectly traced or displaying signal intensity, such as fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense materials, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal gold, or enzymes; for example, fluorescent microspheres, colored latex microspheres, acridinium esters, alkaline phosphatase, horseradish peroxidase, and colloidal gold. In practical use, those skilled in the art can select appropriate solid phases and markers according to detection conditions or actual needs. "Conjugated compounds" typically refer to HIV proteins that are directly or indirectly bound to a solid phase or a marker, such as HIV protein-conjugated compound-solid phase, or HIV protein-conjugated compound-marker. The conjugated compound can be one or more substances, such as commonly used inert proteins (e.g., BSA), biotin, or avidin. In some cases, the conjugated portion can be derivatized. Conjugation can be performed in any manner.

[0053] The term "nucleic acid" as used herein is well known to those skilled in the art and includes DNA (e.g., cDNA) or RNA (e.g., mRNA). Nucleic acids can be double-stranded or single-stranded, linear or circular. The nucleic acid molecule is preferably contained in a vector, which is preferably contained in a host cell. The host cell, after transformation or transfection with the nucleic acid sequence described in this invention, is able to express the binding molecule. To achieve this, the nucleic acid molecule is operatively linked to an element controlling expression.

[0054] The term "vector" as used herein is a tool well-known to those skilled in the art for carrying nucleic acids or genetic material, generally possessing the function of delivering nucleic acids or genetic material to cells. It typically includes, but is not limited to, plasmids, viruses, granules, and artificial chromosomes. Generally, genetically engineered vectors contain an origin of replication, a multiple cloning site, and optional markers. The vector itself is usually a nucleotide sequence, often a DNA sequence, comprising an insert (the target nucleic acid) and a larger sequence serving as the vector's "backbone." Modern vectors, in addition to the target nucleic acid and backbone, may also include promoters, genetic markers, antibiotic resistance, reporter genes, protein purification tags, etc. Vectors can express the target nucleic acid in host cells and typically contain control sequences, such as promoter sequences that initiate transgenic expression.

[0055] "Host cell" is the cell into which the target nucleic acid has been introduced. It should be understood that these terms refer not only to the specific recipient cell, but also to the progeny or potential progeny of these cells. This is because certain modifications may occur in offspring due to mutations or environmental influences, and these offspring may actually differ from the parent cell but are still included within the scope of the terminology used herein.

[0056] "Reagent kit" and "kit" are interchangeable. A kit or reagent does not need to have a box structure, but only needs to be relatively independent and have a suitable loading or container, such as tubes, boxes, bottles, or cards; some components are contained in different containers, and if permitted, some components can be combined in one container.

[0057] The term "antibody" in this article is not particularly limited and may include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, recombinant antibodies, chimeric antibodies, single-chain antibodies, and single-domain antibodies. It may also include functional fragments of the antibodies, such as Fab fragments, F(ab') fragments, Fab'-SH fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv fragments (scFv), dAb fragments, separated complementarity-determining regions (CDRs), and anti-idiotypic antibodies, bifunctional antibodies, or bidomain antibodies.

[0058] The HIV protein of the present invention can be prepared by any suitable method known in the art. For example, in some embodiments, nucleic acid encoding the HIV protein of the present invention can be prepared, cloned into any suitable vector such as plasmid, bacteriophage, or granule, and then expressed by a suitable host through a suitable expression system (e.g., bacterial, yeast, or CHO cell expression system).

[0059] The HIV protein of the present invention allows for mutations at specific locations and allows for the addition of other independent fragments at the N-terminus and / or C-terminus. In some embodiments, the HIV protein has at least one of the following mutations at the corresponding positions of the amino acid sequence shown in SEQ ID NO:1: L30 mutation to M; S83 mutation to N or V; E86 mutation to D or Q; E113 mutation to K or H; N136 mutation to A or S. In some embodiments, mutations occur at C63S and C69S relative to the amino acid sequence shown in SEQ ID NO:1. In some embodiments, a fusion chaperone (e.g., a GST tag) is present at the N-terminus and / or C-terminus of the HIV protein. In some embodiments, the N-terminus and / or C-terminus of the HIV protein is linked to the fusion chaperone via a linker (e.g., (G4S)n). In some embodiments, the N-terminus and / or C-terminus of the HIV protein has an HIV-2 antigenic peptide or an HIV-1 gp120 antigenic peptide. In some embodiments, an HIV-2 antigenic peptide is linked to the N-terminus and / or C-terminus of the HIV protein via a linker (e.g., GGSGG). In some embodiments, the HIV protein is linked to a fusion chaperone and an HIV-2 antigenic peptide (e.g., HIV-2 gp36). The HIV protein is obtained by codon optimization based on its amino acid sequence and using a suitable preparation method.

[0060] In some embodiments, HIV antibodies in samples from subjects recognize epitopes in the HIV protein of the present invention, thus the HIV protein of the present invention can be used in immunoassays to detect HIV antibodies in samples from subjects. In some embodiments, the HIV protein of the present invention can be used for immunoassays, such as ELISA, fluorescence immunochromatography, colloidal gold immunochromatography, chemiluminescence assay, electrochemiluminescence assay, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding assays or methods. In some embodiments, the HIV protein of the present invention can be used as a capture antigen, a detection antigen, or both. In some embodiments, the other antigen paired with the HIV protein of the present invention can be the same or different, as long as it includes fragments of the recombinant HIV antigen of the present invention. For example, in some embodiments, the other antigen paired with the HIV protein of the present invention can be an antigen completely identical to the HIV protein of the present invention, or a different antigen containing corresponding fragments of the HIV protein of the present invention. In some embodiments, such as in an ELISA assay, the HIV protein can be used as a capture antigen coated on a solid phase, such as magnetic beads, to capture HIV antibodies in the sample, and the results are read after color development. In some embodiments, the antigens or antibodies used in the immunoassay can be immobilized on a surface, such as a solid support, like a plastic, membrane such as a nitrocellulose membrane, glass, magnetic beads, or a metal support. In some embodiments, a sample from a subject is contacted with said solid support and then reacted with a detection antibody or detection antigen bearing a detectable label for color development. In this document, the sample from the subject may include biological tissues, cells, or body fluids in a healthy or pathological state, such as blood samples, such as plasma, serum, blood products, such as semen, or vaginal secretions.

[0061] In some embodiments, the detection antigen or detection antibody (such as anti-human IgG antibody or anti-human IgM antibody) can be labeled with a detectable marker. In some embodiments, there are no particular limitations on the detectable marker used to label the antigen or antibody. In some embodiments, the labeling may include, but is not limited to, fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels, as well as indirect labels such as enzymes or ligands, for example, for indirect detection via enzymatic reactions or molecular interactions. In some embodiments, exemplary labels include, but are not limited to, radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / avidin, spin labels, phage labels, and so on.

[0062] In some embodiments, the present invention provides a method, such as an immunoassay for detecting the presence of anti-HIV antibodies in a sample from a subject, the method comprising: contacting the HIV protein of the present invention with the sample; forming a complex of the HIV antibody and the HIV protein in the presence of HIV antibodies in the sample; and detecting the presence of the complex, wherein the presence of the complex indicates the presence of HIV antibodies in the sample. In some embodiments, the complex can be detected by identifying a detectable marker (e.g., a fluorescent marker). In some embodiments, a sample containing or suspected of containing HIV antibodies can be contacted simultaneously or in any order with the HIV protein of the present invention and at least one detection antibody (e.g., a second or third detection antibody, such as an anti-IgG antibody or anti-IgM antibody with a detectable marker). In some embodiments, the method and / or kit of the present invention are suitable for any suitable automated or semi-automated system.

[0063] In some embodiments, the present invention provides a kit comprising the HIV protein of the present invention, such as a kit for detecting the presence of HIV antibodies from a subject sample. In some embodiments, the kit comprises reagents suitable for performing immunoassays. In some embodiments, the kit may comprise instructions for using the immunodiagnostic reagents of the present invention (e.g., conjugates comprising HIV proteins) in an immunoassay for detecting HIV antibodies. In some embodiments, the kit may comprise calibrators or controls, such as standard or control HIV antibodies. In some embodiments, the HIV protein or conjugate of the present invention is contained in a container such as a test tube, microplate, or test strip in the kit. In some embodiments, the kit may also comprise a solid support such as magnetic beads, test tubes, microplates, cuvettes, membranes, filter paper, syringes, pipettes, buffers such as assay buffers, wash buffers, pretreatment reagents, detectable labels such as enzyme-labeled substrate solutions, etc.

[0064] In some embodiments, the present invention includes test strips containing the HIV protein, such as lateral chromatography test strips. In some embodiments, the test strip contains HIV protein coated on a solid phase, at least one detection antibody or at least one detection antigen with a detectable label (such as colloidal gold). In some embodiments, the test strip contains HIV protein with a detectable label, at least one detection antibody or at least one detection antigen coated on a solid phase. In some embodiments, the present invention can rapidly and accurately detect HIV antibodies in a subject by visual inspection or by a fully automated chemiluminescence instrument. In some embodiments, the kit can be prepared using a double-antigen sandwich principle. For example, in some embodiments, antibodies in the sample are captured by HIV protein coated on a solid phase, or antibodies in the sample are detected by HIV protein labeled with a detectable label. In some embodiments, the kit can be prepared using an indirect method principle. For example, in some embodiments, antibodies in the sample are captured by HIV protein coated on a solid phase. In some embodiments, an excitation solution is added, and the luminescence value is measured using a fully automated chemiluminescence instrument. The luminescence value is positively correlated with the total antibody concentration in the sample, and compared with a threshold value to determine whether the result is positive or negative.

[0065] In some implementations, the methods and / or kits described herein can be used to detect the presence of HIV antibodies in samples from subjects, or to measure the amount or concentration of HIV antibodies. Attached Figure Description

[0066] Figure 1 : Illustration of the carrier PE;

[0067] Figure 2 : SDS-PAGE gel images of different HIV proteins without mercaptoethanol; where 1-8 correspond to HIV-F-1-2 to HIV-F-8-2, respectively. Detailed Implementation

[0068] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0069] Design of HIV proteins

[0070] Mutation design was performed on the HIV protein sequence shown in SEQ ID NO:1 to obtain better protein detection performance. After analysis and mutation screening of the HIV protein, the preferred mutation positions were identified as positions 30, 83, 86, 113, and 136 from the N-terminus to the C-terminus, specifically: L30 to M; S83 to N or V; E86 to Q or D; E113 to K or H; and N136 to S or A.

[0071] The specific clone names and corresponding mutations are as follows: HIV-F-1: L30M, S83V, E86D, E113H, N136A; HIV-F-2: L30M, S83V, E86D, E113K, N136S; HIV-F-3: L30M, S83N, E86Q, E113K, N136S; HIV-F-4: L30M, S83N, E86D, E113H, N136S HIV-F-5: L30M, S83V, E86Q, E113H, N136A; HIV-F-6: L30M, S83N, E86Q, E113K, N136A; HIV-F-7: L30M, S83V, E86Q, E113K, N136S; HIV-F-8: L30M, S83N, E86D, E113H, N136A; HIV-F-9: No mutation.

[0072] Design and construction of expression plasmids

[0073] Expression plasmids for HIV-F-1 to HIV-F-9 were constructed. Using the nucleic acid encoding the protein shown in SEQ ID NO:1 as a template, mutant primers were designed using a primer design tool and synthesized by a third-party company. Mutant clones were constructed using site-directed mutagenesis. Simultaneously, a fusion chaperone, GST, was introduced at the N-terminus of the HIV protein. The gene sequence contained restriction enzyme sites BamHI and EcoRI at both ends. The mutant gene was treated with the restriction endonucleases BamHI and EcoRI, respectively. The treated gene fragments were ligated into Phyto's proprietary vector PE (e.g., ...). Figure 1 As shown in the figure, the corresponding expression plasmids were obtained, wherein HIV-F-9 did not require site-directed mutagenesis. Simultaneously, comparisons were made with existing HIV proteins, namely HIV-F-10 (amino acid sequence corresponding to SEQ ID NO:2) and HIV-F-11 (amino acid sequence corresponding to SEQ ID NO:3), and the construction methods were consistent.

[0074] SEQ ID NO:2:

[0075] TLTVQARQLLSGIVQQQRNLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTTVPWNVSWSNKSLSEIWDNMTWMEWEREIANYTKEIYTLIEESQNQQEKNEPELLELDKWASLWNWFD

[0076] SEQ ID NO:3:

[0077] TLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWFDIT

[0078] HIV protein induced expression

[0079] The expression plasmids of HIV-F-1 to HIV-F-11 were transformed into Escherichia coli ER2566(NEB) to obtain the corresponding recombinant Escherichia coli. The recombinant E. coli cells were cultured in LB medium containing 30 μg / ml kanamycin and grown at 37°C until OD600 = 1.5. Then, 1 mM IPTG was added to induce cytoplasmic overexpression at 30°C.

[0080] Four hours after induction, cells were harvested by centrifugation at 7000 rpm for 3 min. The harvested cells were frozen and stored at -20°C. The frozen cells were resuspended in PBS solution and sonicated to disrupt the lysis. The lysate was then centrifuged and filtered. The sample was passed through a GST Focurose 4FF column for elution, and the eluted target protein was stored.

[0081] Preparation of detection reagents

[0082] 1. Coated magnetic beads

[0083] Before coating, the magnetic beads are resuspended and washed, then coated with coating buffer and mixed. Magnetic bead activator EDC / NHS is added, and the mixture is incubated on a 25°C shaker for 30 minutes. After magnetic separation, the beads are washed, and the raw material to be coated is added and mixed. The mixture is incubated on a 25°C shaker for 2.5 hours. After magnetic separation, the beads are washed twice. Blocking buffer (Tris buffer containing BSA, Tween 20, and Proclin 300) is added and vortexed to remove unbound raw material. After magnetic separation, blocking buffer is added, and the mixture is incubated on a shaker for 20-28 hours to block the magnetic bead coating. After magnetic separation, the mixture is washed once with magnetic bead dilution buffer (Tris buffer containing BSA, Tween 20, and Proclin 300) and stored.

[0084] 2. Acridinium ester labeling

[0085] Take the raw material to be labeled, add acridinium ester and mix well, react at 25°C for 60 minutes; add 0.1M glycine to the reactant and mix well; react at 25°C for 30 minutes, place the reactant in buffer solution and dialyze at 4°C, change the dialysis buffer every 2 hours, dialyze 5 times; recover the dialyzed sample to obtain the raw material labeled with acridinium ester.

[0086] 3. HRP tag

[0087] Take HRP and freshly prepared 0.1M NaIO4 solution, stir for 20 minutes at room temperature in the dark, dialyze against 1mM pH 4.4 sodium acetate buffer, incubate overnight at 4°C, adjust pH to 9.0-9.5, then add the raw material to be labeled, and gently stir for 2 hours at room temperature in the dark in 1ml of 0.01M carbonate buffer. Add 0.1ml of freshly prepared 4mg / ml NaBH4 solution, mix well, and incubate at 4°C for 2 hours. Dialyze against 0.15M pH 7.4 PBS, incubate overnight at 4°C, add an equal volume of saturated ammonium sulfate, incubate at 4°C for 1 hour, centrifuge for half an hour, and discard the supernatant. Wash the precipitate twice with semi-saturated ammonium sulfate, and finally dissolve the precipitate in a small amount of 0.15M pH 7.4 PBS. Dialyze against 0.15M pH 7.4 PBS buffer, centrifuge at 10,000 rpm for 30 minutes to remove the precipitate, and the supernatant is the HRP-labeled enzyme conjugate.

[0088] 4. Enzyme immunoassay plate coating

[0089] Dilute the raw material to be coated with 0.05M pH 9.51 CB. Add 100 μL to each well of the polystyrene plate and incubate overnight at 4°C. The next day, discard the solution in the wells, wash once, block with BSA at 37°C for 1 hour, and pat dry for later use.

[0090] Test methods and test results

[0091] 1. Activity detection

[0092] Magnetic beads were coated with HIV-F-1 to HIV-F-11 target proteins, and acridine ester was labeled with another HIV antigen (purchased from Phytok). The solutions were diluted to 0.1 μg / ml to 0.2 μg / ml to prepare working solutions for the magnetic beads and the labeling solution, respectively. 100 μl of the sample to be tested and 50 μl of the magnetic bead working solution were added to the wells of the luminescent plate and reacted at 37 °C for 15 min. Then, the beads were adsorbed by the magnetic plate and washed with 1×PBST solution. 100 μl of the labeling solution was added to the wells of the luminescent plate and reacted at 37 °C for 10 min. The beads were then adsorbed by the magnetic plate again and washed with 1×PBST solution. AE excitation solution was added, and the luminescence value was read.

[0093] The luminescence values ​​of diluted HIV-1 antibody-positive samples (146# / 1K dilution) and blanks were measured, with 3 replicates. The mean (rounded) and P / N (positive / blank ratio, rounded) results are shown in the table below. The detection activity and P / N ratio of HIV-F-1 to HIV-F-8 were significantly improved.

[0094] HIV protein 146# / 1K positive mean blank Average P / N HIV-F-1 481300 563 855 HIV-F-2 458700 547 839 HIV-F-3 440467 566 778 HIV-F-4 423100 563 752 HIV-F-5 402367 588 684 HIV-F-6 374100 630 594 HIV-F-7 395533 521 759 HIV-F-8 418767 550 761 HIV-F-9 231400 518 447 HIV-F-10 41377 349 119 HIV-F-11 109533 294 373

[0095] Chimeric antigen design

[0096] To achieve HIV-2 antibody detection, a gp36 antigenic peptide was chimeric at the C-terminus of HIV proteins (HIV-F-1 to HIV-F-8), with a linker GGSGG between them. Restriction enzyme sites BamHI and EcoRI were placed at both ends of the designed chimeric target gene fragment. The chimeric gene was treated with restriction endonucleases BamHI and EcoRI, respectively. The treated chimeric gene fragment was then ligated into Phypon's proprietary vector PE. The remaining steps followed the design and construction of expression plasmids, with the gp36 antigenic peptide shown in SEQ ID NO:4, yielding chimeric antigens HIV-F-1-1 to HIV-F-8-1.

[0097] Test methods and test results

[0098] 1. HIV-1 and HIV-2 activity detection

[0099] The enzyme immunoassay plate was coated with HIV-F-1-1 to HIV-F-8-1 target proteins, and HRP was labeled with another HIV antigen (purchased from Feipeng). The detection procedure was as follows: 50 μl of the sample to be tested was added and incubated at 37°C for 60 min; the liquid in the wells was discarded, and the plate was washed 5 times with washing buffer, soaking for 30 seconds each time; 100 μl of enzyme conjugate working solution was added to each well and incubated at 37°C for 30 min; washing: the liquid in the wells was discarded, and the plate was washed 5 times with washing buffer, soaking for 30 seconds each time; 50 μl each of chromogenic reagent A and B were added to each well, and the plate was gently shaken to mix, and incubated at 37°C in the dark for 30 min; 50 μl of stop solution was added to each well and the plate was gently shaken to mix; the OD values ​​were read at 450 nm and 630 nm on a microplate reader.

[0100] The values ​​of diluted HIV-1 antibody-positive samples (1# / dilution 30K), HIV-2 positive samples (2# / dilution 1.6K), and blanks were measured. Three replicates were set up, and the mean values ​​(rounded) were calculated. The results are shown in the table below. The chimeric antigens can effectively detect HIV type 1 and type 2.

[0101]

[0102]

[0103] 2. Stability

[0104] Cysteine ​​residues in HIV-F-1-1 to HIV-F-8-1 were mutated to serine residues. Following the aforementioned cloning method, eight new clones, HIV-F-1-2 to HIV-F-8-2, were obtained. The stability of the HIV proteins before and after mutation was assessed by accelerated thermal evaluation at 37°C for 7 days. SDS-PAGE protein gel analysis without β-mercaptoethanol treatment showed no significant changes in the protein structure of HIV-F-1-2 to HIV-F-8-2 after 7 days at 37°C. (See details...) Figure 2 .

[0105] 3. Specificity and sensitivity

[0106] HIV-F-1 to HIV-F-8, HIV-F-1-1 to HIV-F-8-1, and HIV-F-1-2 to HIV-F-8-2 were measured using 1052 negative serum samples, with a specificity of 99.9% for each sample. When measured using 100 positive serum samples, the sensitivity for each sample was 100%.

Claims

1. A protein, wherein a combinatorial mutation is performed at the corresponding position of the amino acid sequence shown in SEQ ID NO:1, wherein the combinatorial mutation is one of the following seven groups of combinatorial mutations: Combination 1: L30M, S83V, E86D, E113H, N136A; Combination 2: L30M, S83V, E86D, E113K, N136S; Combination 3: L30M, S83N, E86D, E113H, N136S; Combination 4: L30M, S83V, E86Q, E113H, N136A; Combination 5: L30M, S83N, E86Q, E113K, N136A; Combination 6: L30M, S83V, E86Q, E113K, N136S; Combination 7: L30M, S83N, E86D, E113H, N136A.

2. The protein according to claim 1, further subjected to one of the following mutations at the corresponding position of the amino acid sequence shown in SEQ ID NO:1: C63S, C69S.

3. The protein according to any one of claims 1-2, wherein the N-terminus and / or C-terminus further comprises an HIV-2 antigenic peptide.

4. The protein according to any one of claims 1-2, wherein its N-terminus and / or C-terminus also have a fusion chaperone.

5. The protein according to any one of claims 1-2, wherein the N-terminus and / or C-terminus further comprises a linker.

6. A conjugate comprising the protein and conjugate portion as described in any one of claims 1-5.

7. The conjugate according to claim 6, wherein the conjugate portion is selected from solid phases, markers, or chelates.

8. A nucleic acid for encoding the protein of any one of claims 1-5.

9. A kit comprising the protein of any one of claims 1-5 or the conjugate of any one of claims 6-7.

10. Use of the protein of any one of claims 1-5 or the conjugate of any one of claims 6-7 in the preparation of an HIV antibody detection kit.

11. A detection method for non-diagnostic purposes, comprising contacting a sample with the protein of any one of claims 1-5, the conjugate of any one of claims 6-7, or the kit of claim 9.

12. The conjugate according to claim 7, wherein the solid phase is selected from microspheres, plates, and membranes.

13. The conjugate according to claim 7, wherein the solid phase is selected from magnetic microspheres, plastic microspheres, latex microparticles, microporous plates, glass, capillaries, nylon or nitrocellulose membranes.

14. The conjugate according to claim 7, wherein the marker is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes.

15. The conjugate according to claim 7, wherein the marker is selected from fluorescent microspheres, colored latex microspheres, acridine esters, alkaline phosphatase, horseradish peroxidase, or colloidal gold.

16. The conjugate according to claim 7, wherein the conjugate is selected from inert proteins, biotin, or avidin.

17. A vector comprising the nucleic acid of claim 8.

18. A host cell comprising the vector of claim 17.

19. The kit of claim 9, further comprising other components mixed with the protein or conjugate.

20. The kit according to claim 9, wherein the kit further comprises additional HIV protein.

21. The kit according to claim 9, wherein the kit further comprises a secondary antibody.

22. The kit according to claim 21, wherein the secondary antibody is an anti-human antibody.

23. The detection method according to claim 11, wherein the detection method is used to detect HIV antibodies in a sample.

Images