Synthetic peptides for HIV-1 detection

Abstract

The present invention relates to the field of medical diagnosis, and more specifically, to a synthetic peptide for detecting HIV-1. The synthetic peptide is derived from the amino acid sequence of SEQ ID NO:1 by mutation. It enables better detection of anti-HIV-1 antibodies and can better avoid potential "false negative" and "false positive" results.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis, in particular to a synthetic peptide for detecting HIV-1. Background Art

[0002] Since its discovery in the 1980s, AIDS, caused by the human immunodeficiency virus (HIV), has spread globally. According to the World Health Organization, by the end of 2016, there were approximately 36.7 million people living with HIV worldwide. As of June 30, 2017, there were 718,000 people living with HIV / AIDS in my country. HIV primarily attacks the body's helper T lymphocyte system, selectively targeting cells bearing CD4 molecules, damaging the immune system and causing it to lose its defenses. This often leads to serious opportunistic infections, which in turn can lead to various diseases and cancers, ultimately leading to AIDS. To date, there are no effective drugs or vaccines to treat or prevent AIDS, making HIV infection one of the most serious infectious diseases and public health threats globally.

[0003] HIV is a lipid-enveloped RNA retrovirus. Its single-copy RNA gene is approximately 9.2-9.8 kilobases (kb) long. HIV has three main structural genes: env, gag, and pol, each encoding a protein or antigen that produces a different function. After HIV enters the body, antibodies typically develop within about six weeks. Antibodies to the HIV precursor protein p55 and the core protein p24 appear first in the serum, followed by antibodies to the outer envelope protein GP120 and the transmembrane glycoprotein gp41. Currently, the main HIV strains identified are HIV-1 and HIV-2. There are four known strains of HIV: M, N, O, and P. M and N are the most widespread. To date, only two cases of P subtype have been reported worldwide, and O subtype has only 100,000 cases, primarily concentrated in Central and West Africa. These subtypes are biologically similar, with genomic homology of 40%-50%. Currently, HIV-1 is the most prevalent in my country.

[0004] Due to the high costs of instruments, reagents, and labor required for HIV antigen and nucleic acid testing, HIV antibody testing remains the dominant method in the HIV testing market. Currently, HIV antibody testing materials are being developed to improve sensitivity and specificity, shorten the window period, and be simpler and faster. To meet these development trends, there is a need in the field to provide HIV-1 recombinant proteins that can be used for HIV-1 antibody testing.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention generally relates to methods and materials for diagnosing human immunodeficiency virus (HIV) infection, and more particularly to synthetic peptides having reactive properties that bind to HIV-1 antibodies and their uses. The present invention is intended to provide diagnostic laboratories with a means, specifically a specific peptide, that allows for better detection of anti-HIV-1 antibodies and better avoids potential "false negative" and "false positive" results.

[0007] The synthetic peptide is the amino acid sequence shown in SEQ ID NO: 1 including the mutation site;

[0008] The mutation site is selected from at least one of the following:

[0009] T77 substitution to V, N, or S;

[0010] N101 is replaced by D, E or Q;

[0011] G105 is substituted with K, H, or R;

[0012] P124 is replaced by D or E;

[0013] K127 is replaced by D, E or Q;

[0014] Or, an amino acid sequence having at least 80% sequence identity with any of the above sequences, and having a sensitivity greater than 99% and / or a specificity greater than 99.4%.

[0015] Compared with the prior art, the synthetic peptide provided by the present invention can achieve a sensitivity of 100% and a specificity of over 99.7% through one or more amino acid mutation sites. DETAILED DESCRIPTION

[0016] The present invention relates to a synthetic peptide, which is an amino acid sequence shown in SEQ ID NO: 1 and includes a mutation site;

[0017] The mutation sites are selected from 1, 2, 3, 4 or 5 of the following:

[0018] T77 substitution to V, N, or S;

[0019] N101 is replaced by D, E or Q;

[0020] G105 is substituted with K, H, or R;

[0021] P124 is replaced by D or E;

[0022] K127 is replaced by D, E or Q;

[0023] or an amino acid sequence having at least 80%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity to any of the above sequences;

[0024] In some embodiments, the synthetic peptides, kits or detection methods provided herein have a sensitivity greater than 99%, greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, or greater than 99.9%.

[0025] In some embodiments, the synthetic peptides, kits, or detection methods provided herein have a specificity greater than 99.4% (e.g., greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, greater than 99.9%, or higher).

[0026] The sensitivity and specificity referred to above are determined according to the invention in the Examples of the present invention.

[0027] In some embodiments, the present invention provides a polypeptide comprising all or part of the sequence shown in SEQ ID NO: 1, for example, at least 30 consecutive amino acids.

[0028] The synthetic peptides provided by the present invention are preferably purified and are particularly suitable for the identification and / or enrichment of HIV-1 viruses in animals, particularly mammals, particularly primates, and particularly humans. HIV-1 retrovirus is the most common and is prevalent in many regions of the world.

[0029] In some embodiments, the mutation site includes: T77 is replaced by V, N or S.

[0030] In some embodiments, the mutation site includes: N101 is replaced by D, E or Q.

[0031] In some embodiments, the mutation site includes: G105 is replaced by K, H or R.

[0032] In some embodiments, the mutation site includes: P124 is replaced by D or E.

[0033] In some embodiments, the mutation site includes: K127 is replaced by D, E or Q.

[0034] In some embodiments, the mutation site further comprises 1, 2, 3, 4 or 5 of the following mutation sites:

[0035] R18 is replaced by S;

[0036] Q95 is replaced by E;

[0037] L106 was replaced by E;

[0038] L123 was replaced by K;

[0039] N130 is replaced by K.

[0040] In some embodiments, the mutation site includes: R18 is replaced by S.

[0041] In some embodiments, the mutation site includes: Q95 is replaced by E.

[0042] In some embodiments, the mutation site includes: L106 is replaced by E.

[0043] In some embodiments, the mutation site includes: L123 is replaced by K.

[0044] In some embodiments, the mutation site includes: N130 is replaced by K.

[0045] In some embodiments, the mutation site is:

[0046] R18 is substituted with S; T77 is substituted with V, N or S; Q95 is substituted with E; N101 is substituted with D, E or Q; G105 is substituted with K, H or R; L106 is substituted with E; L123 is substituted with K; P124 is substituted with D or E; K127 is substituted with D, E or Q; N130 is substituted with K.

[0047] In some embodiments, the mutation site is:

[0048] T77 is substituted with V; Q95 is substituted with E, or not substituted; N101 is substituted with D, E or Q; G105 is substituted with K, H or R; L106 is substituted with E, or not substituted; P124 is substituted with D or EK127 is substituted with D, E or Q.

[0049] In some embodiments, the mutation sites are: R18 is replaced by S, or not replaced; T77 is replaced by V, N or S; Q95 is replaced by E, or not replaced; N101 is replaced by D, E or Q; G105 is replaced by K, H or R; L106 is replaced by E, or not replaced; L123 is replaced by K, or not replaced; P124 is replaced by D or E; K127 is replaced by D, E or Q; N130 is replaced by K, or not replaced.

[0050] In some embodiments, the mutation sites are: R18 is replaced by S; T77 is replaced by S; Q95 is replaced by E, or not replaced; N101 is replaced by D, E or Q; G105 is replaced by K, H or R; L106 is replaced by E, or not replaced; L123 is replaced by K; P124 is replaced by D; K127 is replaced by D, E or Q; N130 is replaced by K.

[0051] In some embodiments, the present invention provides an article comprising the synthetic peptide, such as a microtiter plate (on which the synthetic peptide may be coated).

[0052] The present invention also relates to an isolated nucleic acid molecule encoding a synthetic peptide as described above.

[0053] The present invention also relates to a vector comprising the nucleic acid molecule described above.

[0054] The present invention also relates to a host cell transformed with the vector described above.

[0055] The host cell may be a eukaryotic cell, such as a mammalian cell.

[0056] The present invention also relates to a method for producing a synthetic peptide as described above, comprising:

[0057] a) expressing a nucleic acid molecule as described above; or

[0058] b) Chemical synthesis, where amino acids are added to obtain the complete polypeptide.

[0059] By the above-mentioned synthesis method a), the expressed polypeptide can be enriched by affinity purification using well-known methods.

[0060] Chemical synthesis can be performed using equipment known to those skilled in the art, such as an automatic peptide synthesizer, such as the automatic peptide synthesizer sold by Applied BioSystems, to obtain the peptide through synthesis.

[0061] The present invention also relates to a modified synthetic peptide obtained by labeling the synthetic peptide described above with an indicator showing signal intensity.

[0062] In some embodiments, the indicator showing signal intensity comprises any one of a fluorescent substance, quantum dots, digoxigenin-labeled probe, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal gold, or enzymes.

[0063] In some embodiments, the fluorescent substance includes Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluorescein, rare earth metal cryptates, europium trisbipyridyldiamine, europium cryptates or chelates, diamines, bis-anthocyanin, La Jolla blue dye, allophycocyanin, allococyanin B, any one of phycocyanin C, phycocyanin R, thiamine, phycoerythrin, phycoerythrin R, REG, rhodamine green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas Red.

[0064] In some embodiments, the radioisotope comprises 110 In, 111 In, 177 Lu, 18 F. 52 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 86 Y. 90 Y. 89 Zr, 94 mTc, 94 Tc, 99 mTc, 120 I. 123 I. 124 I. 125 I. 131 I.154-158 Gd, 32 P. 11 C. 13 N. 15 O. 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br, 76 Br, 82 mRb and 83 Any one of Sr.

[0065] In some embodiments, the enzyme includes any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

[0066] In some embodiments, the fluorescent microspheres are polystyrene fluorescent microspheres encapsulated with rare earth fluorescent ion europium.

[0067] The present invention also relates to a kit comprising the synthetic peptide as described above, or the modified synthetic peptide as described above.

[0068] In some embodiments, the kit further comprises one or more of an immunologically acceptable diluent, a buffer, a protease inhibitor, a blocking agent for blocking non-specific binding of antibodies, and a second antibody having affinity for HIV-1 antibodies.

[0069] In some embodiments, the protease inhibitors can be selected from one or more of PMSF, EDTA, NaN3, pepstantin, leupeptin, aprotinin, Indinavir, Ritonavir, Nelfinavir, Amprenavir, and Kaletra.

[0070] In some embodiments, the second antibody having affinity for HIV-1 antibodies can be an anti-human Fc region antibody, and the species can be mouse, rat, rabbit, dog, sheep, horse or human;

[0071] In some embodiments, the second antibody having affinity for HIV-1 antibody is labeled with the above-mentioned indicator for displaying signal intensity.

[0072] In some embodiments, the blocking agent for blocking non-specific binding of antibodies comprises BSA and FBS.

[0073] In some embodiments, the buffer is phosphate buffered saline (PBS), Tris buffered saline (TBS), Tris buffered saline-Tween (TBST), or Tris buffered saline-Triton (TBST).

[0074] In some embodiments, the kit further comprises a microtiter plate, such as described above.

[0075] In some embodiments, when the kit includes the modified synthetic peptide as described above, the kit further includes a reagent for detecting the indicator showing signal intensity;

[0076] The reagents used to detect the indicator showing the signal intensity are well known to those skilled in the art. As an example, when the indicator is horseradish peroxidase (HRP), the corresponding reagents can be hydrogen peroxide and luminol; when the indicator is biotin, the corresponding reagent can be avidin, and so on.

[0077] The present invention also relates to a method for detecting anti-HIV-1 antibodies, which comprises contacting a biological sample with the synthetic peptide as described above, or the modified synthetic peptide as described above, or the reagents in the kit as described above to form an immune complex;

[0078] detecting the presence of the synthetic peptide or the modified synthetic peptide in the immune complex to indicate the presence of HIV-1 antibodies in the biological sample;

[0079] The methods are performed for diagnostic or non-diagnostic purposes, in vivo or in vitro.

[0080] The present invention also relates to the use of the synthetic peptide, the modified synthetic peptide, or the kit described above in the preparation of HIV-1 diagnostic agents.

[0081] In some embodiments, provided herein are methods for obtaining or designing a recombinant protein for detecting HIV, which may include one or more of the following steps:

[0082] Select the human immunodeficiency virus envelope protein, select some segments of the protein, and perform mutation design on some sites. Prepare the corresponding recombinant protein through expression and purification. Then, obtain a recombinant protein with high activity and good specificity through activity and specificity screening.

[0083] Synthesize the sequence fragment and connect it to a vector such as pMD18-T vector to construct a T vector containing the target fragment;

[0084] Through site-directed mutagenesis design, construct a vector containing a specific mutation target fragment, such as the pMD18-T vector;

[0085] Preparation of recombinant protein: Design upstream primers (e.g., with an EcoR I restriction site) and downstream primers (e.g., with a BamH I restriction site). Using a vector containing the target fragment, such as a T vector, as a template, amplify the target gene. After double enzyme digestion and purification, ligate the target gene into a vector, such as pET-28a, and transform cells, such as Escherichia coli BL21. After screening for positive clones, pick a single colony and inoculate it into a culture medium, such as LB medium containing 50ul / ml Kan, and culture it with shaking at an appropriate temperature, such as 37 degrees. After the OD600 reaches 0.6-0.8, add 1.0 mM IPTG, induce the culture at 37 degrees for 2-4 hours, extract the total protein, and identify the expression of the recombinant protein by SDS-PAGE. Purify the target protein for the first time using an NI ion chelation column and the second time using an SP column. The purity of the recombinant protein is identified by SDS-PAGE.

[0086] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0087] Example 1

[0088] Design of recombinant proteins

[0089] The gene sequence encoding SEQ ID NO: 1 was synthesized by gene synthesis, and the amino acids at positions X77 (X77 represents the 77th position of the sequence shown in SEQ ID NO: 1, the same below), X95, X101, X105, X106, X124, and X127 were mutated.

[0090] The amino acid at position X77 is mutated from T to V, N or S, the amino acid at position X95 is mutated from Q to E, the amino acid at position X101 is mutated from N to E, D or Q, the amino acid at position X105 is mutated from G to K, H or R, the amino acid at position X106 is mutated from L to E, the amino acid at position X124 is mutated from P to D or E, and the amino acid at position X127 is mutated from K to D, E or Q.

[0091] Mutant clones were constructed according to the above mutation directions. The preferred scheme is that X77, X101, X105, X124, and X127, which are V, E, K, D, and D, respectively, are named HIV-Ag-1; N, D, H, E, and E are named HIV-Ag-2; S, Q, R, D, and Q are named HIV-Ag-3; S, D, R, E, and E are named HIV-Ag-4; V, D, K, E, and D are named HIV-Ag-5; N, E, H, D, and E are named HIV-Ag-6; V, Q, R, D, and Q are named HIV-Ag-7; S, Q, K, E, and D are named HIV-Ag-8; and N, Q, H, E, and Q are named HIV-Ag-9. These clones were then constructed into the pMD18-T vector (TaKara Dalian Takara Biotechnology, Cat. No. 6011). The gene sequence of SEQ ID NO: 1 was constructed and named pMD18-T-HIV-Ag-0, and the mutant clones were named pMD18-T-HIV-Ag-1 to pMD18-T-HIV-Ag-9 for subsequent amplification and nucleic acid fragment storage.

[0092] Example 2

[0093] Design of recombinant proteins

[0094] Based on Example 1, mutations were designed for the amino acids in sequences X18, X95, X106, X123, and X130 in HIV-Ag-1 to HIV-Ag-9.

[0095] The amino acid at position X18 was mutated from R to S, the amino acid at position X95 was mutated from Q to E, the amino acid at position X106 was mutated from L to E, the amino acid at position X123 was mutated from L to K, and the amino acid at position X130 was mutated from N to K.

[0096] The new sequences were named HIV-Ag-10 to HIV-Ag-18 and constructed into the pMD18-T vector, named pMD18-T-HIV-Ag-10 to pMD18-T-HIV-Ag-18, for subsequent amplification and nucleic acid fragment storage.

[0097] Example 3

[0098] Construction, induction and purification of recombinant protein expression vectors

[0099] Construction of recombinant protein expression vector and inducible expression: Upstream primers (with EcoRI restriction sites) and downstream primers (with BamHI restriction sites) were designed, and the target gene was amplified using pMD18-T-HIV-Ag-0 to pMD18-T-HIV-Ag-18 as templates. After purification, the target gene was double-digested with EcoRI (TaKara Dalian Takara Biotechnology, Catalog No. 1010A) and BamHI restriction endonucleases (TaKara Dalian Takara Biotechnology, Catalog No. 1040A) and incubated at 37°C for 2 hours. The digested product was purified and ligated into the pET-28a vector that had been digested with the same enzymes. The cells were incubated at 22°C for 2 hours and then at 16°C for 2 hours. The ligation product was transformed into competent Escherichia coli BL21 (NEB (New England Biolabs), Cat. No. C2530H) using the heat shock method and plated onto LB plates containing 50 μg / ml Kan. The cells were cultured at 37°C for 16 hours. Positive clones were selected, identified by PCR and double enzyme digestion, and then sent for sequencing. Single positive clones that were sequenced correctly were inoculated into LB medium containing 50 μg / ml Kan and cultured at 37°C with shaking. 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 hours. Total protein was extracted and recombinant protein expression was analyzed by SDS-PAGE. The recombinant protein, with its N-terminal 6*HIS tag, was purified by nickel ion chelation and then purified by SP column to obtain a protein with a purity of 96%. The resulting proteins were designated HIV-Ag-0 to HIV-Ag-18.

[0100] Example 4

[0101] Application of mutant cloned HIV-Ag-1 to HIV-Ag-9 recombinant antigens in enzyme immunoassay product technology and evaluation of activity and specificity

[0102] The enzyme immunoassay was performed as follows:

[0103] Coating: Add the recombinant protein to 50mM CB pH 9.6 coating solution at a working concentration of 100ng / ml. Mix well and add 100ul per well to the polystyrene plate. Coat at 4℃ for 18-20 hours.

[0104] Blocking: Remove the coated plate and equilibrate at room temperature for 30 minutes. Wash twice with detergent and add 150 μl of blocking solution to each well for 2 hours at 37°C. Pat dry and place in an electronic drying oven with a humidity of less than 30% for 24 hours before use.

[0105] Labeling: Label the recombinant protein with HRP according to the recommended method, dilute to 50 ng / ml in enzyme working solution, and mix thoroughly (enzyme diluent formula: 20 mM PB, 150 mM NaCl, 0.5% BSA, 0.05% Tween-200, 0.1% P300).

[0106] Reaction mode and reaction time: 50 ul of the sample to be tested + 50 ul of sample diluent, react in a constant temperature box at 37 degrees for 60 minutes; wash the plate 5 times, pat dry, add 100 ul of labeled recombinant protein-HRP working solution, react at 37 degrees for 30 minutes; wash the plate 5 times, add 50 ul each of color developer A and B, and develop for 30 minutes; add 50 ul of stop solution, use dual wavelength detection at 450 nm and 630 nm, and the detection should be completed within 10 minutes.

[0107] A performance comparison experiment was conducted with a commercially available kit using 500 HIV-1 positive sera and 3,000 HIV-1 antibody negative sera. The results are shown in Table 1. The recombinant protein of the present invention has improved sensitivity and specificity for HIV-1 antibody detection, outperforming existing products.

[0108] Table 1 Detection results after mutation

[0109] Number of positive HIV samples detected in 500 samples (sensitivity) Number of HIV-negative samples detected (specificity / false positive rate) Control reagent (commercially available) 499(99.8%) 18（99.40% / 0.60%） HIV-Ag-0 498（99.6%） 24（99.20% / 0.80%） HIV-Ag-1 500（100%） 6（99.80% / 0.20%） HIV-Ag-2 500（100%） 8（99.73% / 0.27%） HIV-Ag-3 500（100%） 8（99.73% / 0.27%） HIV-Ag-4 500（100%） 5（99.83% / 0.17%） HIV-Ag-5 500（100%） 7（99.77% / 0.23%） HIV-Ag-6 500（100%） 6（99.80% / 0.20%） HIV-Ag-7 500（100%） 7（99.77% / 0.23%） HIV-Ag-8 500（100%） 9（99.70% / 0.30%） HIV-Ag-9 500（100%） 9（99.70% / 0.30%）

[0110] Example 5

[0111] Activity evaluation of recombinant antigens from mutant cloned HIV-Ag-1 to HIV-Ag-18 in enzyme immunoassay

[0112] The enzyme immunoassay was performed as follows:

[0113] Coating: Add the recombinant protein to 50mM CB pH 9.6 coating solution at a working concentration of 100ng / ml. Mix well and add 100ul per well to the polystyrene plate. Coat at 4℃ for 18-20 hours.

[0114] Blocking: Remove the coated plate and equilibrate at room temperature for 30 minutes. Wash twice with detergent and add 150 μl of blocking solution to each well for 2 hours at 37°C. Pat dry and place in an electronic drying oven with a humidity of less than 30% for 24 hours before use.

[0115] Labeling: Label the recombinant protein with HRP according to the recommended method, dilute to 50 ng / ml in enzyme working solution, and mix thoroughly (enzyme diluent formula: 20 mM PB, 150 mM NaCl, 0.5% BSA, 0.05% Tween-200, 0.1% P300).

[0116] Reaction mode and reaction time: 50 ul of the sample to be tested + 50 ul of sample diluent, react in a constant temperature box at 37 degrees for 60 minutes; wash the plate 5 times, pat dry, add 100 ul of labeled recombinant protein-HRP working solution, react at 37 degrees for 30 minutes; wash the plate 5 times, add 50 ul each of color developer A and B, and develop for 30 minutes; add 50 ul of stop solution, use dual wavelength detection at 450 nm and 630 nm, and the detection should be completed within 10 minutes.

[0117] 500 HIV-1 positive sera were diluted 1:200 with sample diluent and used as test specimens for activity testing. The results are shown in Table 2. After the mutation, the average sensitivity of the diluted 500 positive specimens was significantly improved.

[0118] Table 2 Detection results after mutation

[0119] Clone Name Average OD600 reading of 500 HIV samples diluted 1:200 HIV-Ag-1 0.732 HIV-Ag-10 0.856 HIV-Ag-2 0.794 HIV-Ag-11 1.122 HIV-Ag-3 0.642 HIV-Ag-12 0.989 HIV-Ag-4 1.254 HIV-Ag-13 1.261 HIV-Ag-5 0.901 HIV-Ag-14 1.101 HIV-Ag-6 1.264 HIV-Ag-15 1.367 HIV-Ag-7 0.892 HIV-Ag-16 1.299 HIV-Ag-8 0.697 HIV-Ag-17 1.189 HIV-Ag-9 1.103 HIV-Ag-18 1.179

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. SEQUENCE LISTING <110> Dongguan Pengzhi Biotechnology Co., Ltd. <120> Synthetic peptides for HIV-1 detection <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 139 <212> PRT <213> Human Immunodeficiency Virus <400> 1 Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln 1 5 10 15 Gln Arg Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln 20 25 30 Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala Val 35 40 45 Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser 50 55 60 Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp Asn Thr Ser Trp Ser 65 70 75 80 Asn Lys Ser Leu Ser Glu Ile Trp Asp Asn Met Thr Trp Met Gln Trp 85 90 95 Glu Arg Glu Ile Asn Asn Tyr Thr Gly Leu Ile Tyr Thr Leu Ile Glu 100 105 110 Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Leu Pro Leu Leu Lys Leu 115 120 125 Asp Asn Trp Ala Ser Leu Trp Asn Trp Phe Asp 130 135

Claims

1. A synthetic peptide, characterized in that It is based on the amino acid sequence shown in SEQ ID NO: 1 and simultaneously carries out any one of the following mutations 1) to 9): 1) X77, X101, X105, X124, and X127 mutated to V, E, K, D, and D, respectively; 2) X77, X101, X105, X124, and X127 mutated to N, D, H, E, and E, respectively; 3) X77, X101, X105, X124, and X127 mutated to S, Q, R, D, and Q, respectively; 4) X77, X101, X105, X124, and X127 mutated to S, D, R, E, and E, respectively; 5) X77, X101, X105, X124, and X127 mutated to V, D, K, E, and D, respectively; 6) X77, X101, X105, X124, and X127 mutated to N, E, H, D, and E, respectively; 7) X77, X101, X105, X124, and X127 mutated to V, Q, R, D, and Q, respectively; 8) X77, X101, X105, X124, and X127 mutated to S, Q, K, E, and D, respectively; 9) X77, X101, X105, X124, and X127 mutated to N, Q, H, E, and Q, respectively.

2. An isolated nucleic acid molecule, characterized in that It encodes the synthetic peptide according to claim 1.

3. A carrier, characterized in that It comprises the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that It is transformed with the vector according to claim 3.

5. A method for producing the synthetic peptide according to claim 1, characterized in that: include: a) expressing the nucleic acid molecule according to claim 2; or, b) Chemical synthesis, where amino acids are added to obtain the complete polypeptide.

6. A modified synthetic peptide, characterized in that The method is obtained by labeling an indicator showing signal intensity with the synthetic peptide according to claim 1.

7. The modified synthetic peptide according to claim 6, characterized in that The indicator showing signal intensity includes any one of fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent markers, ultrasound contrast agents, photosensitizers, colloidal gold or enzymes.

8. A kit, characterized in that It includes the synthetic peptide according to claim 1 or the modified synthetic peptide according to claim 6 or 7.

9. The kit according to claim 8, characterized in that The composition may further comprise one or more of an immunologically acceptable diluent, a buffer, a protease inhibitor, a blocking agent for blocking non-specific binding of the antibody, and a second antibody having affinity for the HIV-1 antibody.

10. The kit according to claim 8, characterized in that When the kit comprises the modified synthetic peptide according to claim 6 or 7, the kit further comprises a reagent for detecting the indicator showing signal intensity.

11. A method for detecting anti-HIV-1 antibodies in vitro for non-diagnostic purposes, characterized in that: The method comprises contacting a biological sample with the synthetic peptide according to claim 1, or the modified synthetic peptide according to claim 6 or 7, or the reagent in the kit according to any one of claims 8 to 10 to form an immune complex; The presence of the synthetic peptide or the modified synthetic peptide in the immune complex is detected to indicate the presence of HIV-1 antibodies in the biological sample.

12. Use of the synthetic peptide according to claim 1, or the modified synthetic peptide according to claim 6 or 7, or the kit according to any one of claims 8 to 10 in the preparation of an HIV-1 diagnostic agent.