HIV gp41 polypeptide mutants and uses thereof

Abstract

The application provides an HIV gp41 polypeptide mutant and application thereof, and relates to the technical field of biology.The amino acid sequence of the HIV gp41 polypeptide mutant comprises the amino acid sequence of the HIV gp41 polypeptide in which the 122th and / or 124th amino acid is a non-charged amino acid.The HIV gp41 polypeptide mutant has improved sensitivity and specificity in detecting HIV-1 antibodies in a sample, and improves the accuracy of HIV antibody detection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an HIV gp41 polypeptide mutant and its applications. Background Technology

[0002] The following statements are provided only as background information in relation to the present invention and do not necessarily constitute prior art.

[0003] Acquired immunodeficiency syndrome is a disease caused by infection with the human immunodeficiency virus (HIV). The HIV virus 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 (encapsulated by the p7 nucleocapsid protein), reverse transcriptase, integrase, proteases, 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 (mainly 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 or gp125, and gp41 or gp36, forming the gp120-gp41 or gp125-gp36 complex active protein.

[0004] Currently, there are two types of HIV: HIV-1 and HIV-2. HIV-1 is the primary cause of ADIS (Adverse Events of the Disease). 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. There are currently eight known types of HIV-2, but only two types cause large-scale epidemics: group A and group B.

[0005] HIV targets the human immune system. Without any treatment, the immune system of an HIV-infected person will be gradually destroyed by HIV until they lose almost all their immune capacity. The main modes of HIV transmission include blood transmission, mother-to-child transmission, and sexual transmission. HIV antibody testing is one of the main methods for screening for HIV infection. HIV proteins are important raw materials for HIV antibody testing and are related to the sensitivity and specificity of the test.

[0006] gp41 is the envelope glycoprotein of HIV-1 and one of the main targets for HIV-1 antibody detection. The detection sensitivity and specificity of gp41 are crucial for HIV-1 antibody detection. gp41 is highly hydrophobic and carries many charged amino acids, making it prone to non-specific reactions with other proteins through hydrophobic interactions and electrostatic adsorption, leading to false positives. HIV antibody detection requires extremely high specificity. Therefore, designing a suitable gp41 region and optimizing its amino acid sequence are essential for improving the accuracy of HIV antibody detection. Summary of the Invention

[0007] The inventors of this application have conducted extensive research on the gp41 membrane glycoprotein and, considering its hydrophilicity / hydrophobicity and the charge of its amino acid residues, constructed a mutant HIV gp41 polypeptide. The purpose of this application is to provide the HIV gp41 polypeptide mutant and its applications.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0009] In a first aspect, an HIV gp41 polypeptide mutant is provided, wherein the amino acid sequence of the HIV gp41 polypeptide mutant includes an uncharged amino acid at position 122 and / or position 124, and the position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO:1.

[0010] In a second aspect, a method for preparing an HIV gp41 polypeptide mutant is provided, the method comprising replacing the charged amino acid at position 122 and / or position 124 of the amino acid sequence of the original HIV-1 gp41 polypeptide with a non-charged amino acid.

[0011] Thirdly, a conjugate is provided, the conjugate comprising the HIV gp41 polypeptide mutant and the conjugate portion described in the first aspect.

[0012] Fourthly, a biomaterial is provided, said biomaterial comprising polynucleotides, carriers, or cells;

[0013] (i) a polynucleotide, said polynucleotide encoding the HIV gp41 polypeptide mutant described in the first aspect;

[0014] (ii) a vector carrying the polynucleotide in (i);

[0015] (iii) Cells carrying the polynucleotides described in (i), or containing the vector described in (ii), or expressing the HIV gp41 polypeptide mutant described in the first aspect.

[0016] Fifthly, a kit is provided comprising the HIV gp41 polypeptide mutant described in the first aspect, or the conjugate described in the third aspect.

[0017] In a sixth aspect, the use of the HIV gp41 polypeptide mutant described in the first aspect, or the conjugate described in the third aspect, or the kit described in the fourth aspect, in detecting HIV-1 antibodies, diagnosing HIV infection, or identifying HIV-1 infection is provided.

[0018] In a seventh aspect, a method for detecting anti-HIV-1 antibodies in a sample is provided, the method comprising: contacting the sample to be tested with the HIV gp41 polypeptide mutant described in the first aspect, the conjugate described in the third aspect, or the kit described in the fifth aspect to form an immune complex;

[0019] Based on the detection results of the complex, it is determined whether HIV-1 antibodies are present in the sample. Detailed Implementation

[0020] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0022] In this document, unless otherwise stated, any numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbers I, III; i, ii, iii; X1 to X4, first, second to fifth, etc.

[0023] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0024] In this document, the terms “comprising” or “including” mean that the stated elements, integers or steps are included, but do not exclude any other elements, integers or steps.

[0025] In this article, "each...independently selected" and "...independently selected" are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0026] In this article, "HIV gp41 polypeptide" refers to the gp41 subunit that makes up the HIV-1 envelope glycoprotein. The HIV-1 envelope glycoprotein is composed of heterodimers of the receptor-binding protein gp120 and the transmembrane protein gp41, which form a trimer and are distributed on the viral membrane surface. In this article, "original HIV gp41 polypeptide" refers to the HIV gp41 polypeptide with position 122 and / or position 124 of the sequence not replaced with an uncharged amino acid. "Original HIV gp41 peptide" can be a wild-type HIV gp41 peptide, such as HIV gp41 peptides of M, N, O, or P subtype HIV-1, or HIV gp41 peptides of subtypes A, B, C, D, E, F, G, H, I, J, K, or L; "Original HIV gp41 peptide" can also be an artificially modified HIV gp41 peptide, including peptides or proteins that have been substituted, truncated, or fused with other domains.

[0027] In this document, peptides, polypeptides, and proteins are not strictly distinguished and may be used interchangeably in some cases. Generally, peptides refer to polymers composed of amino acids linked by peptide bonds, whether naturally occurring or synthetic. Polypeptides may also contain non-amino acid components, such as carbohydrate groups, metal ions, or carboxylic acid esters. Non-amino acid components may be added by the cells expressing the polypeptide and may vary depending on the cell type. Polypeptides are defined in this document with respect to their amino acid backbone structure or the nucleic acid encoding them. The addition of carbohydrate groups is generally not specified, but is permissible. All polypeptide sequences are written according to generally accepted conventions, with the α-N-terminal amino acid residue on the left and the α-C-terminal amino acid residue on the right. When used herein, the term "N-terminus" refers to the free α-amino group of an amino acid in a polypeptide, and the term "C-terminus" refers to the free α-carboxylic acid terminus of an amino acid in a polypeptide. A polypeptide ending with a group at the N-terminus refers to a polypeptide carrying a group on the α-amino nitrogen of an N-terminal amino acid residue. An amino acid ending with a group at the N-terminus refers to an amino acid carrying a group on its α-amino nitrogen.

[0028] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), 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).

[0029] In this document, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when two sequences are optimally aligned. The alignment of amino acid sequence identity percentages can be performed using various methods within the art, such as software well-known in the field, including BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA. Those skilled in the art can determine appropriate parameters for the aligned sequences, including any algorithms required to achieve maximum alignment of the full length of the compared sequences. Unless otherwise specified, differences between any two sequences include, but are not limited to, insertions, deletions, or mutations.

[0030] In this document, the term "having at least 90% identity" can mean having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity.

[0031] Unless otherwise specified, amino acid positions in this document refer to the amino acid sequence shown in SEQ ID NO:1. However, it should be noted, and those skilled in the art will understand, that different sequences can have different numbering systems, for example, if additional amino acid residues are added or removed compared to SEQ ID NO:1. Therefore, when referring to a specific amino acid residue by its number, this description is not limited to the amino acid precisely located at that numbered position when counting from the amino acid sequence of SEQ ID NO:1, but also refers to the equivalent / corresponding amino acid residue in any and all sequences, the equivalent / corresponding position of which can be obtained by comparison in accordance with the above definition of "identity," even if the residue is not at the same precise numbered position. For example, if the sequence is shorter or longer than SEQ ID NO:1, or has insertions or deletions compared to SEQ ID NO:1. For example, if sequence X is missing 2 amino acid residues at the N-terminus compared to the sequence of SEQ ID NO:1, then position 1 of sequence X is equivalent / corresponding to position 3 of the sequence of SEQ ID NO:1. When not specifically indicated that the amino acid position in sequence X is used as a reference, the referred third amino acid residue refers to position 1 when the amino acid position in sequence X is used as a reference.

[0032] Based on the polarity of the R group in their side chains, amino acids can be classified into nonpolar amino acids, polar uncharged amino acids, negatively charged amino acids, and positively charged amino acids. Nonpolar amino acids are those whose side chains are nonpolar or hydrophobic groups. Examples of nonpolar amino acids include, but are not limited to, alanine, valine, leucine, isoleucine, tryptophan, proline, phenylalanine, and methionine. Polar uncharged amino acids are those whose side chains are non-dissociated polar groups (or hydrophilic groups). These amino acids can form hydrogen bonds with water. Examples of polar uncharged amino acids include, but are not limited to, glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine. Negatively charged amino acids are those whose side chains are negatively charged after dissociation; they are also called acidic amino acids. Examples of negatively charged amino acids include, but are not limited to, aspartic acid and glutamic acid. Positively charged amino acids are those that carry a positive charge at pH 7; they are also called basic amino acids. Examples of positively charged amino acids include, but are not limited to, lysine, arginine, and histidine. In this article, "charged amino acids" refers to negatively charged and positively charged amino acids that meet the above definition; "non-charged amino acids" refers to non-polar and polar non-charged amino acids that meet the above definition, wherein "non-charged amino acids" are preferably polar non-charged amino acids.

[0033] In this document, the term "conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue that is physicochemically similar, such that the substitution does not alter or substantially alters the properties and function of the entire polypeptide or protein. Conservative amino acid substitution is well known to those skilled in the art. Families of amino acid residues with similar side chains are known in the art, such as substitutions between nonpolar amino acids, between polar uncharged amino acids, between negatively charged amino acids, and between positively charged amino acids as defined above. Specific forms of conserved amino acid substitution include those that are replaced with amino acids not among the normal 20 amino acids encoded by the genetic code. Embodiments of the present invention may use synthetic peptides, and thus such "non-naturally occurring" amino acid residues may be used in the peptides disclosed herein, and the natural saturated carbon chains in the side chains of amino acid residues may be exchanged for shorter or longer saturated carbon chains.

[0034] In this document, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, including ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, it may optionally encode the sense or antisense strand. Nucleic acid molecules can be naturally occurring, synthetic, recombinant, or any combination thereof. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" are used interchangeably.

[0035] In this document, the term "vector" refers to a delivery system that can operatively insert a genetic element (such as the aforementioned nucleic acid molecule) into and express that genetic element, for example, to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. Vectors can be used to transform, transduce, or transfect host cells, enabling the expression of the genetic element they carry within the host cells. Examples of vectors include: plasmids, episome plasmids, microcircular DNA, phage particles, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), bacteriophages such as λ phage or M13 phage, and animal viruses. Vectors may contain various elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. Vectors may also include components that facilitate their entry into the cell, including but not limited to viral particles, liposomes, or protein coats. Vectors can be expression vectors or cloning vectors. In some embodiments, the vectors (e.g., expression vectors) provided in this disclosure contain a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in this disclosure, at least one promoter operatively linked to the nucleic acid sequence (e.g., SV40, CMV, EF1α), and at least one selection marker.

[0036] In this document, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Progeny may not be identical to primary cells due to natural, accidental, or intentional mutations, and may differ from primary cells morphologically and / or in genomic DNA. “Transformant” and “transformed cell” include primary test cells and cultures derived therefrom. Cells may be prokaryotic or eukaryotic, with prokaryotic cells including, but not limited to, Escherichia coli, Bacillus, or Staphylococcus. Eukaryotic cells include, but are not limited to, mammalian cells, insect cell lines, plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.

[0037] In this article, the term "solid phase" can refer to a substance that can be suspended or dispersed in a liquid phase (e.g., solid carriers such as particles and magnetic beads), or a solid phase that can contain or carry a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic paths, glass capillaries, nanopillars, and monolithic columns).

[0038] In this article, the term "marker" refers to a class of substances that have properties that can be directly observed by the naked eye or detected or probing by instruments, such as luminescence, color development, radioactivity, etc., which enable qualitative or quantitative detection of the corresponding target.

[0039] In a first aspect, an HIV gp41 polypeptide mutant is provided, wherein the amino acid sequence of the HIV gp41 polypeptide mutant includes an uncharged amino acid at position 122 and / or position 124, and the position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO:1.

[0040] In an optional embodiment, the uncharged amino acid is a polar uncharged amino acid.

[0041] In an optional embodiment, the amino acid sequence of the HIV gp41 polypeptide mutant includes an HIV gp41 polypeptide amino acid sequence with Q at position 122 and / or N at position 124.

[0042] In an optional embodiment, the amino acid sequence of the HIV gp41 peptide mutant has at least 90% identity with the original HIV gp41 peptide amino acid sequence, wherein the original HIV gp41 peptide amino acid sequence is derived from M-type, N-type, O-type, or P-type HIV-1; or, the original HIV gp41 peptide amino acid sequence is derived from subtypes A, B, C, D, E, F, G, H, I, J, K, or L of HIV-1.

[0043] In an optional embodiment, the HIV gp41 polypeptide mutant amino acid sequence contains the amino acid sequence shown in SEQ ID NO:1, or contains an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0044] In an optional embodiment, a conserved amino acid substitution is performed on the amino acid sequence shown in SEQ ID NO:1 compared to the amino acid sequence shown in SEQ ID NO:1, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0045] In optional embodiments, the HIV gp41 peptide mutant in any of the above embodiments also satisfies the requirement that the specificity of HIV-1 antibodies in the detection sample is higher than that of peptides having the amino acid sequences shown in SEQ ID NO:2-12. That is, in detecting the same number of samples, using the HIV gp41 peptide mutant to detect samples produces fewer false positives compared to using peptides having the amino acid sequences shown in SEQ ID NO:2-12.

[0046] In an optional embodiment, the HIV gp41 peptide mutant in any of the above embodiments also satisfies the requirement that the specificity of HIV-1 antibody in the detection sample is higher than 99%.

[0047] In optional embodiments, the HIV gp41 polypeptide mutant in any of the above embodiments also satisfies the requirement that the specificity of the HIV-1 antibody in the detection sample is higher than 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

[0048] In an optional embodiment, the amino acid sequence of the HIV gp41 polypeptide mutant is shown in SEQ ID NO:1.

[0049] The HIV gp41 polypeptide mutant provided in the first aspect improves the sensitivity or specificity to HIV-1 antibodies and HIV-positive samples by optimizing the selected gp41 region of the HIV gp41 protein and optimizing the amino acid sequence.

[0050] In a second aspect, a method for preparing the HIV gp41 polypeptide mutant described in the first aspect is provided, the method comprising replacing the charged amino acid at position 122 and / or position 124 of the amino acid sequence of the original HIV-1 gp41 polypeptide with a non-charged amino acid.

[0051] In an optional embodiment, the preparation method includes replacing position 122 of the original HIV gp41 polypeptide amino acid sequence with Q; or / and replacing position 124 with N.

[0052] In an optional embodiment, the original HIV gp41 polypeptide amino acid sequence is derived from M-type, N-type, O-type, or P-type HIV-1; or, the original HIV gp41 polypeptide amino acid sequence is derived from subtypes A, B, C, D, E, F, G, H, I, J, K, or L of HIV-1.

[0053] In an optional embodiment, the original HIV gp41 polypeptide amino acid sequence contains the amino acid sequence shown in SEQ ID NO:12.

[0054] In an optional embodiment, the preparation method includes culturing cells expressing the HIV gp41 polypeptide mutant, and then isolating and purifying the HIV gp41 polypeptide mutant.

[0055] In an optional embodiment, the preparation method further includes converting and expressing a polynucleotide encoding the HIV gp41 polypeptide mutant into cells.

[0056] In an optional embodiment, the preparation method further includes synthesizing a polynucleotide containing the gene encoding the HIV gp41 polypeptide mutant as needed, and / or preparing a suitable expression vector as needed, transforming the expression vector into the desired cells and expressing it, and obtaining the HIV gp41 polypeptide mutant through purification.

[0057] In an optional embodiment, the HIV gp41 polypeptide mutant is expressed by prokaryotic cells.

[0058] In an optional embodiment, the HIV gp41 polypeptide mutant is expressed by Escherichia coli.

[0059] The preparation method provided in the second aspect only requires mutation to obtain the HIV gp41 polypeptide mutant described in the first aspect, and it can be expressed using a mature expression system in the field, thus having high versatility.

[0060] Thirdly, a conjugate is provided, the conjugate comprising the HIV gp41 polypeptide mutant and the conjugate portion described in the first aspect.

[0061] In an optional embodiment, the joining portion is selected from solid phase, marker, or overseas Chinese association material.

[0062] In an optional embodiment, the solid phase is selected from microspheres, plates, and membranes.

[0063] In an optional embodiment, the solid phase is selected from magnetic microspheres, plastic microspheres, latex microparticles, microporous plates, glass, capillaries, nylon or nitrocellulose membranes.

[0064] In optional embodiments, the label is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes.

[0065] In an optional embodiment, the marker is selected from fluorescent microspheres, colored latex microspheres, acridine esters, alkaline phosphatase, horseradish peroxidase, or colloidal gold.

[0066] In practical use, those skilled in the art can select appropriate markers according to the detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this application.

[0067] In an optional implementation, the compound is selected from inert proteins, biotin, or avidin.

[0068] Fourthly, a biomaterial is provided, said biomaterial comprising polynucleotides, carriers, or cells;

[0069] (i) a polynucleotide, said polynucleotide encoding the HIV gp41 polypeptide mutant described in the first aspect;

[0070] (ii) a vector carrying the polynucleotide in (i);

[0071] (iii) Cells carrying the polynucleotides described in (i), or containing the vector described in (ii), or expressing the HIV gp41 polypeptide mutant described in the first aspect.

[0072] Fifthly, a kit is provided comprising the HIV gp41 peptide mutant described in the first aspect, or the conjugate described in the third aspect. This kit uses the HIV gp41 peptide mutant or a conjugate containing it as the antigen for binding HIV antibodies, thereby improving the sensitivity or specificity of the kit's detection.

[0073] In an optional implementation, the kit is used for immunoassay and contains reagents for immunoassay.

[0074] In optional embodiments, the immunoassay includes chemiluminescence detection, immunochromatographic detection, ELISA detection, immunomagnetic particle detection, immunofluorescence detection, or immunoblotting detection.

[0075] In optional embodiments, the kit includes reagents and / or consumables for detection, including but not limited to one or more of primers, probes, buffers, dyes, diluents, washing solutions, chromogenic solutions, lysis buffers, negative controls, positive controls, and blank controls. Those skilled in the art can select the reagent composition of the kit according to the specific detection method, and this invention does not limit this selection.

[0076] In this article, "kit" and "reagent" are used interchangeably. Kits or reagents do not need to have a box structure; they only require relative independence and suitable loading or containers, such as tubes, boxes, bottles, or cards. Some components may be placed in different containers, while others may be combined into one container if permissible.

[0077] In an optional embodiment, the kit may further include molecules that provide other HIV antigenic epitopes, including but not limited to molecules of one or more of the following antigenic epitopes: gp120 antigen, gp160 antigen, gp125 antigen, and gp36 antigen.

[0078] In an optional implementation, the kit may further include a p24 antibody.

[0079] In an optional embodiment, the kit includes an immunochromatographic test strip, wherein the HIV gp41 polypeptide mutant is coated on the immunochromatographic test strip; the immunochromatographic test strip is further coated with an HIV gp41 second antigen, and the HIV gp41 polypeptide mutant is used to capture the complex formed by the binding of the second antigen and the HIV gp41 antibody.

[0080] In an optional embodiment, the HIV gp41 polypeptide mutant is coated on the detection line, and the second antigen is linked to the marker.

[0081] In a sixth aspect, the use of the HIV gp41 polypeptide mutant described in the first aspect, the conjugate described in the third aspect, or the kit described in the fifth aspect in detecting HIV-1 antibodies, diagnosing HIV infection, or identifying HIV-1 infection is provided.

[0082] In a seventh aspect, a method for detecting anti-HIV-1 antibodies in a sample is provided, the method comprising: contacting the sample to be tested with the HIV gp41 polypeptide mutant described in the first aspect, the conjugate described in the third aspect, or the kit described in the fifth aspect to form an immune complex;

[0083] Based on the detection results of the complex, it is determined whether HIV-1 antibodies are present in the sample.

[0084] In an optional implementation, the method for detecting anti-HIV-1 antibodies in the sample is not for diagnostic or therapeutic purposes.

[0085] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0086] Example 1: Construction of recombinant protein expression plasmid containing HIV gp41 polypeptide mutant

[0087] In this embodiment, the following amino acid sequence encoding the HIV gp41 polypeptide mutant (as shown in Table 1) was used for cloning optimization and construction. The corresponding gene fragments can be obtained by gene synthesis, template amplification or bridge PCR. The gene fragments and expression vectors are digested and ligated by restriction endonucleases, T4 DNA ligase and other tool enzymes to construct expression plasmids.

[0088] Table 1. Amino acid sequences encoding the HIV gp41 polypeptide mutant.

[0089]

[0090]

[0091] Example 2: Induction and purification of HIV gp41 polypeptide mutant

[0092] HIV gp41 peptide mutant expression induction: The constructed expression plasmid was transformed into *E. coli* expression strains using a heat shock method and plated on LB agar plates containing 100 μg / mL kanamycin, incubated at 37°C for 16 h. Colonies were picked, and positive strains identified by bacterial culture PCR were sequenced. After successful sequencing, the strains were inoculated into LB agar plates containing 50 μg / mL kanamycin and incubated with shaking at 37°C. When the OD600 reached 0.6–0.8, 1.0 mM IPTG was added, and the culture was induced at 37°C for 2–4 h. SDS-PAGE was used to identify the expression of the HIV gp41 peptide mutant. The purified HIV gp41 peptide mutants are shown in Table 1.

[0093] Example 3: Evaluation of the sensitivity and specificity of recombinant HIV antigen using a double-antigen sandwich method on a colloidal gold chromatography platform.

[0094] The purified HIV recombinant antigen was evaluated using a colloidal gold chromatography platform, as detailed below:

[0095] 1. HIV gp41 recombinant antigen labeling: Take 5 mL of 40,000 / 10,000 colloidal gold, add an appropriate amount of 0.2 M K2CO3, stir for 5 min, add the HIV gp41 recombinant antigen prepared above, stir for 5 min, and then add an appropriate amount of 10% BSA blocking terminator; centrifuge at 10,000 rpm for 10 min, remove the supernatant, reconstitute the precipitate with gold reconstitution solution, and finally adjust the volume to 0.5 mL (i.e., 1 / 10 of the colloidal gold solution volume) with gold reconstitution solution; finally dilute the labeled antigen concentrated gold by a certain factor with gold reconstitution solution to prepare gold working solution, and spread the gold; freeze-dry the spread gold in a freeze dryer (1-2 h) or dry it overnight in a drying room at 37℃.

[0096] 2. HIV antigen coating: Dilute HIV antigen (purchased from Feipeng Biotechnology Co., Ltd.) to 2.0 mg / mL with coating diluent, coat with the antigen, and incubate at 37°C for 1-2 hours.

[0097] 3. Preparation of gold bar: The gold bar is cut into strips of the required width using a strip cutter, assembled, and then sampled for testing.

[0098] 4. Application of Colloidal Test Strips: During testing, the antibody first binds to the colloidal gold-labeled antigen to form an antibody-colloidal gold-labeled antigen complex. Due to capillary action, the antibody-colloidal gold-labeled antigen complex migrates forward along the nitrocellulose membrane. Upon reaching the test line, the antibody-colloidal gold-labeled antigen complex binds to the streaked coating antigen, forming an antigen-antibody-colloidal gold-labeled antigen complex, which accumulates on the test line, forming a red precipitate line. The intensity of the red precipitate line indicates the strength of the reaction; the deeper the red, the stronger the reactivity, and vice versa. The strength of the reaction is represented by the letter C and a number, with smaller numbers after C indicating stronger reactivity. The absence of a red precipitate line is indicated by B. The above colloidal gold-labeled test strips are used to test quality control samples and clinical negative samples.

[0099] 5. Test Results: The color rendering level was determined using a color chart, and the results are as follows:

[0100] Table 2

[0101]

[0102] The results showed that I270, I270A, I270B, I270E, I270F, I270G, I270H, I270I, I270J, or I270K had better activity than I270C or I270D, and I270K had enhanced specificity.

[0103] Furthermore, I270K antigen and control antigen were used to amplify and detect clinical positive and negative samples, respectively. Clinical HIV-I positive samples (numbers 1-50) collected from the hospital for Roche testing showed that I270K antigen had improved detection sensitivity compared to the control antigen; some results are shown in Table 3. Testing 1000 clinical negative samples showed that I270K antigen had improved detection specificity compared to the control antigen, with a specificity of 99.9% (1 / 1000), significantly superior to the control antigen.

[0104] Table 3. Results of magnified detection of clinical positive samples

[0105]

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An HIV gp41 polypeptide mutant, characterized in that, The amino acid sequence of the HIV gp41 polypeptide mutant includes an uncharged amino acid at position 122 and / or position 124, and the position of the amino acid residue is determined with reference to the amino acid sequence shown in SEQ ID NO:

1.

2. The HIV gp41 polypeptide mutant according to claim 1, characterized in that, The uncharged amino acids are polar uncharged amino acids; Optionally, the amino acid sequence of the HIV gp41 polypeptide mutant includes an HIV gp41 polypeptide amino acid sequence with Q at position 122 and / or N at position 124.

3. The HIV gp41 polypeptide mutant according to claim 1 or 2, characterized in that, The amino acid sequence of the HIV gp41 peptide mutant has at least 90% identity with the original HIV gp41 peptide amino acid sequence, which is derived from M, N, O, or P type HIV-1; or, the original HIV gp41 peptide amino acid sequence is derived from A, B, C, D, E, F, G, H, I, J, K, or L subtype HIV-1. Optionally, the HIV gp41 polypeptide mutant amino acid sequence contains the amino acid sequence shown in SEQ ID NO:1, or contains an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:

1. Optionally, the specificity of the HIV-1 antibody in the HIV gp41 peptide mutant detection sample is higher than 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

4. A method for preparing an HIV gp41 polypeptide mutant, characterized in that, The preparation method includes replacing the charged amino acid at position 122 and / or position 124 in the amino acid sequence of the original HIV-1gp41 peptide with a non-charged amino acid. Optionally, the preparation method includes replacing E at position 122 with Q in the original HIV gp41 polypeptide amino acid sequence; or / and replacing D at position 124 with N; Optionally, the original HIV gp41 polypeptide amino acid sequence is derived from M, N, O, or P type HIV-1; or, the original HIV gp41 polypeptide amino acid sequence is derived from A, B, C, D, E, F, G, H, I, J, K, or L subtype HIV-1. Optionally, the original HIV gp41 polypeptide amino acid sequence contains the amino acid sequence shown in SEQ ID NO:

12.

5. A conjugate, characterized in that, Includes the HIV gp41 polypeptide mutant and conjugated portion as described in any one of claims 1 to 3; Optionally, the joining portion is selected from solid phases, markers, or overseas Chinese association materials; Optionally, the label is selected from fluorescent substances, quantum dots, digoxigenin-labeled probes, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers, colloidal metals, or enzymes. Alternatively, the Overseas Chinese Federation may select inert proteins, biotin, or avidin.

6. A biomaterial, characterized in that, Including polynucleotides, carriers, or cells; (i) a polynucleotide, said polynucleotide encoding the HIV gp41 polypeptide mutant according to any one of claims 1 to 3; (ii) a vector carrying the polynucleotide in (i); (iii) A cell carrying the polynucleotide of (i), or containing the vector of (ii), or expressing the HIV gp41 polypeptide mutant of any one of claims 1 to 3.

7. A reagent kit, characterized in that, It includes the HIV gp41 polypeptide mutant according to any one of claims 1 to 3, or the conjugate according to claim 5.

8. The use of the HIV gp41 polypeptide mutant according to any one of claims 1 to 3, or the conjugate according to claim 5, or the kit according to claim 7 in detecting HIV-1 antibodies, diagnosing HIV infection, or identifying HIV-1 infection.

9. A method for detecting anti-HIV-1 antibodies in a sample, characterized in that, include: The HIV gp41 polypeptide mutant according to any one of claims 1 to 3, the conjugate according to claim 5, or the kit according to claim 7 are brought into contact with the sample to be tested to form an immune complex; Based on the detection results of the complex, it is determined whether HIV-1 antibodies are present in the sample.

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