Human immunodeficiency virus recombinant antigen and preparation method and application thereof

Abstract

The application relates to the technical field of biology, and discloses a human immunodeficiency virus recombinant antigen and a preparation method and application thereof, the antigen has the structure shown in formula (1): NH2-FP-NHR-X1-CHR-MPER-L1-FP-NHR-X2-CHR-MPER-L2-FP-NHR-X3-CHR-MPER-COOH formula (1); wherein one or two of X1, X2 and X3 are each independently a Loop sequence, and the rest are each independently a connecting peptide; wherein L1 and L2 are each independently a connecting peptide; wherein FP and MPER are each independently none, a partial sequence or a complete sequence. The human immunodeficiency virus recombinant antigen provided by the application can be realized soluble expression or partial soluble expression, and after purification, can be stable in a conventional physiological buffer solution without a denaturing agent or a surfactant and has good immunoreactivity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing human immunodeficiency virus recombinant antigen, nucleic acid, vector, cell, and application of human immunodeficiency virus recombinant antigen. Background Technology

[0002] Currently, the double-antigen sandwich immunoassay is the most widely used method for immunoassay of human immunodeficiency virus (HIV). In this method, the quality of the labeled antigen is one of the key factors determining the detection sensitivity. The immunogenicity of the labeled antigen directly affects the overall performance of the method. Highly active labeled antigens require good solubility and correct spatial folding conformation, because the immunogenicity of an antigen is highly dependent on its native conformation, especially conformation-dependent epitopes.

[0003] Existing recombinant antigen technologies can be broadly categorized into two types: One approach involves expressing the recombinant antigen as inclusion bodies and then dissolving the recombinant protein using a surfactant. For example, Abbott's patent US8785596 uses 4% SDS to dissolve the gp41 recombinant protein and preserves it in a buffer solution containing 0.1% SDS. If the antigen is expressed as an inclusion body, it usually indicates that it has not folded correctly, resulting in poor solubility; such antigens often have significantly reduced immunogenicity after labeling.

[0004] Another type is the expression of fusion recombinant proteins, which is used in most technical solutions. For example, Phytobio used HT series fusion proteins in patent CN101475642 to solublely express the gp41 recombinant protein; while Roche used fusion proteins such as FkpA, SplA, and SlyD in patents such as US 6962982 and US20090291892 to express soluble recombinant antigens. In terms of antigen reactivity, soluble expression of fusion antigens is generally higher than that of antigens dissolved by surfactants; however, the introduction of fusion proteins increases the risk of nonspecific reactions.

[0005] In summary, the key problem with existing recombinant antigen technologies lies in the difficulty of obtaining soluble recombinant antigens of human immunodeficiency virus. Summary of the Invention

[0006] The purpose of this invention is to overcome the problem of difficulty in obtaining solublely expressible recombinant human immunodeficiency virus antigens in existing technologies, and to provide a recombinant human immunodeficiency virus antigen, its preparation method, and its applications. The recombinant human immunodeficiency virus antigen provided by this invention can achieve soluble or partially soluble expression, and after purification, it is stable and exhibits good immunoreactivity in conventional physiological buffer solutions free of denaturants or surfactants.

[0007] To achieve the above objectives, the first aspect of the present invention provides a recombinant antigen of human immunodeficiency virus having the structure shown in formula (1): NH2-FP-NHR-X1-CHR-MPER-L1-FP-NHR-X2-CHR-MPER-L2-FP-NHR-X3-CHR-MPER-COOH formula (1); Among them, one or two of X1, X2 and X3 are each independently a loop sequence, and the rest are each independently a linking peptide; Among them, L1 and L2 are each independently a linker peptide; Among them, FP and MPER can be either none, a partial sequence, or a complete sequence, respectively.

[0008] Preferably, the loop sequence includes the loop sequence of the gp41 or gp36 protein.

[0009] Preferably, the antigen has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0010] A second aspect of the present invention provides a nucleic acid encoding the recombinant antigen of the human immunodeficiency virus described in the first aspect.

[0011] Preferably, the nucleic acid has the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4.

[0012] A third aspect of the present invention provides a carrier containing the nucleic acid described in the second aspect.

[0013] A fourth aspect of the present invention provides a cell containing the carrier described in the third aspect.

[0014] The fifth aspect of the present invention provides a method for preparing recombinant human immunodeficiency virus antigen, the method comprising: inducing expression in the cells described in the fourth aspect to obtain recombinant human immunodeficiency virus antigen.

[0015] The sixth aspect of this invention provides the use of the human immunodeficiency virus recombinant antigen described in the first aspect, or the nucleic acid described in the second aspect, or the vector described in the third aspect, or the cell described in the fourth aspect in the preparation of human immunodeficiency virus recombinant antigen and / or the preparation of human immunodeficiency virus detection products.

[0016] The seventh aspect of the present invention provides a human immunodeficiency virus (HIV) detection product, the product comprising at least one of the following: the recombinant HIV antigen described in the first aspect, and / or the nucleic acid described in the second aspect, the vector described in the third aspect, and the recombinant HIV antigen prepared from cells described in the fourth aspect.

[0017] Through the above technical solution, the present invention can achieve the following technical effects: The recombinant human immunodeficiency virus antigen provided by this invention can achieve soluble or partially soluble expression, and after purification, it can be stable in conventional physiological buffer solutions without denaturants or surfactants and has good immunoreactivity. Attached Figure Description

[0018] Figure 1 This is a diagram showing the purification process of the HIV1 recombinant antigen in Example 1; Figure 2 This is an electrophoresis diagram of the expression detection of HIV2 recombinant antigen in Example 2. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below. It should be noted that the specific embodiments are only a detailed description of the present invention and should not be regarded as a limitation of the present invention.

[0020] According to publicly available literature in this field, the sequence structure of the extracellular region of gp41 or gp36 protein (gp41 will be used as a substitute hereafter due to the similarity between gp41 and gp36) can be: NH2-FP-NHR-Loop-CHR-MPER-TM-CP-COOH. Here, FP stands for Fusion Peptide, NHR for N-terminal Heptad Repeat, Loop for Loop Region (a disulfide-bonded immunodominant loop region), CHR for C-terminal Heptad Repeat, MPER for Membrane-Proximal External Region, TM for Transmembrane Domain, and CP for Cytoplasmic Domain (sometimes also referred to as Cytoplasmic Tail, CT). The above sequence region division and the function of each region are general knowledge in this field and will not be elaborated here.

[0021] Based on the above-mentioned extracellular region sequence structure of natural proteins, the inventors of this invention proposed a novel structure as shown in formula (1), and unexpectedly discovered that the recombinant antigen of human immunodeficiency virus with this structure can be solublely expressed.

[0022] In this invention, soluble expression can be determined by detecting the expression of recombinant antigens. For example, cells induced to express are taken, lysed, and centrifuged to obtain the supernatant. If the target recombinant antigen is present in the supernatant, it indicates that the recombinant antigen is solublely expressed. As an example, in Examples 1 and 2, the target protein was obtained by purifying the supernatant, meaning the target recombinant antigen was present in the supernatant, indicating that the recombinant antigens in Examples 1 and 2 are solublely expressed. The inventors of this invention further discovered that the key to achieving soluble expression of the human immunodeficiency virus recombinant antigen of this invention lies in: the arrangement of the loop sequence and the design of the linker peptide.

[0023] The first aspect of the present invention provides a recombinant antigen of human immunodeficiency virus having the structure shown in formula (1): NH2-FP-NHR-X1-CHR-MPER-L1-FP-NHR-X2-CHR-MPER-L2-FP-NHR-X3-CHR-MPER-COOH Formula (1); Among them, one or two of X1, X2 and X3 are each independently a loop sequence, and the rest are each independently a linking peptide; Among them, L1 and L2 are each independently a linker peptide; Among them, FP and MPER can be either none, a partial sequence, or a complete sequence, respectively.

[0024] In this invention, the loop sequence refers to the original loop sequence of the human immunodeficiency virus (HIV) protein. Since the structures of many HIV proteins have been resolved, regions can be divided based on known areas or in a known manner to obtain the original loop sequence of the HIV protein. Alternatively, known sequences in the art can be used directly, such as those listed in the Los Alamos HIV Sequence Database (accessible at www.hiv.lanl.gov).

[0025] In this invention, the sequences of the FP, NHR, CHR, and MPER regions can be known sequences in the art. Since the structures of various human immunodeficiency virus proteins have been resolved, regions can be partitioned based on known regions or in a known manner to obtain usable sequences of the FP, NHR, CHR, and MPER regions. Alternatively, known sequences in the art can be used directly, such as the sequences of the FP, NHR, CHR, and MPER regions described in the literature *Biochemistry and Biophysics of HIV-1 gp41membrane interactions*. This literature is cited in its entirety here.

[0026] As an example, the following provides a set of sequences for FP, NHR, CHR, and MPER regions.

[0027] As an example, the sequence for the FP region could be AVGIGALFLGFL.

[0028] As an example, the sequence for the NHR region could be STMGARSMTLTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARIL.

[0029] As an example, CHR WNHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQ.

[0030] As an example, the sequence for the MPER region could be NEQELLELDKWASLWN.

[0031] In this invention, FP and MPER can be deleted, truncated based on known sequences, or retained entirely. Deletion or truncation of FP and MPER is a common practice in the field of HIV viral proteins. Deletion, truncation, or retention of FP and MPER does not affect the soluble expression effect of the recombinant human immunodeficiency virus antigen of this invention.

[0032] In some embodiments, the loop sequence comprises the loop sequence of the gp41 or gp36 protein.

[0033] In some embodiments, the linker peptide has a sequence length of 3-10 amino acids, preferably 5-7 amino acids.

[0034] In some embodiments, the linker peptide comprises GGSSG and / or GGGGS.

[0035] As an example, the present invention provides two specific recombinant antigens, namely HIV1 recombinant antigen (the amino acid sequence of which is shown in SEQ ID NO:1) and HIV2 recombinant antigen (the amino acid sequence of which is shown in SEQ ID NO:3).

[0036] In some embodiments, the antigen has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3.

[0037] In the present invention, SEQ ID The amino acid sequence shown in NO:1 is: MTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRA IEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS.

[0038] In the present invention, SEQ ID The amino acid sequence shown in NO:3 is: MLATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAGGSGGNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQKGSSGGTLLAGIVQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIE KYLKDQAQLNSWGCAFRQVCHTSVPWVNDTLTPDWNNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQKGSSGGTLLAGIVQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAGGSGGNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQKLNS. Compared to existing inclusion bodies, the human immunodeficiency virus recombinant antigen provided by the first aspect of the present invention has the characteristic of soluble expression, the production process is less complex than that of inclusion bodies, and avoids the use of high concentration denaturing agents (such as 8M urea, 7M guanidine hydrochloride).

[0039] Compared to existing fusion proteins, the human immunodeficiency virus recombinant antigen provided in the first aspect of this invention can avoid the non-specific binding caused by fusion proteins during use.

[0040] A second aspect of the present invention provides a nucleic acid encoding the recombinant antigen of the human immunodeficiency virus described in the first aspect.

[0041] It is well known in the art that of the 20 different amino acids that make up proteins, except for Met (ATG) or Trp (TGG), which are encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic codon, most amino acids are determined by more than one codon. The substitution of the third nucleotide in a triplet codon usually does not change the amino acid composition; therefore, genes encoding the same protein can have different nucleotide sequences.

[0042] As an example, the present invention provides a nucleic acid encoding the HIV1 recombinant antigen of the first aspect (the nucleotide sequence of which is shown in SEQ ID NO:2), and a nucleic acid encoding the HIV2 recombinant antigen of the first aspect (the nucleotide sequence of which is shown in SEQ ID NO:4).

[0043] In some embodiments, the nucleic acid has the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4.

[0044] In the present invention, the nucleotide sequence set forth in SEQ ID NO: 2 is: ATGACCGTGCAAGCGCGCCAACTGCTGAGCGGCATCGTTCAACAGCAGAACAACCTGCTGCGTGCGATTGAAGCGCAGCAGCATTTACTGCAGCTGACGGTGTGGGGCATTAAACAACTGCAAGCGCGCATTCTGGCGGTGGAGCGCTATCTGAAAGATCAGCAGCTGCTGGGCATTTGGGGCTGCAGCGGCAAACTGATTTGCACCACCGCGGTGCCGTGGAACGCGAGCTGGAGCAACAAAAGCCTGGAACAGATTTGGAACAACATGACGTGGATGGAATGGGATCGTGAAATTAACAACTATACGAGCCTGATTCATAGCCTGATCGAAGAGAGCCAAAATCAGCAAGAAAAAAATGAGCAAGAATTACTGGAAGGCAGTAGCGGCGGTCAGTTATTAAGCGGTATCGTGCAGCAACAGAATAACTTACTGCGCGCCATTGAAGCGCAGCAACATCTGCTGCAACTGACCGTTTGGGGTATCAAACAGTTACAAGCGCGTATTCTGGCGGGCGGTAGTGGCGGCCATACCACGTGGATGGAGTGGGATCGTGAGATTAATAACTATACGAGCCTGATCCATAGTTTAATTGAAGAAAGTCAGAATCAGCAAGAAAAGAATGAACAAGAATTATTAGAAGGTAGCAGCGGCGGCCAACTGTTAAGCGGCATTGTGCAGCAGCAAAATAACCTGCTGCGCGCGATCGAAGCGCAGCAGCATCTGCTGCAGCTGACCGTTTGGGGTATTAAGCAGCTGCAAGCGCGCATTCTGGCGGGTGGCAGCGGTGGCCATACGACGTGGATGGAATGGGACCGCGAAATCAATAACTACACGAGCTTAATTCATAGCCTGATTGAAGAGAGTCAAAATCAGCAAGAAAAAAACGAGCAAGAACTGCTGGAGTTAGATAAATGGGCGAGC.

[0045] In the present invention, the nucleotide sequence set forth in SEQ ID NO: 4 is: ATGCTAGCGACAGCTGGGTCAGCAATGGGAGCGGCAAGCCTGACCCTGAGCGCACAGTCTCGTACCCTGCTGGCGGGCATCGTGCAGCAGCAACAACAGCTGCTGGACGTTGTTAAACGTCAGCAGGAGATGTTACGTCTGACCGTTTGGGGCACCAAGAACCTGCAGGCGCGTGTAACTGCGGGTGGTTCGGGTGGCAACATGACCTGGCAAGAGTGGGAGCAGAAGGTGCGTTACCTGGAGGCGAATATCAGCCAGAGCTTGGAGCAGGCGCAGATTCAGCAAGAGAAGAACATGTACGAACTGCAGAAGGGTTCCAGCGGTGGCACCCTGTTGGCCGGTATCGTCCAGCAACAACAACAATTGTTGGACGTCGTGAAGCGCCAGCAAGAAATGCTGCGTTTGACGGTCTGGGGTACGAAAAACCTGCAGGCTCGCGTGACCGCGATTGAAAAATATTTAAAAGATCAGGCTCAGTTGAACAGCTGGGGGTGCGCATTCCGCCAGGTTTGTCACACCAGCGTTCCGTGGGTTAATGATACCCTGACTCCGGATTGGAATAATATGACCTGGCAGGAGTGGGAACAAAAGGTACGTTATTTGGAAGCGAACATCTCCCAGTCACTGGAACAGGCGCAAATCCAACAAGAGAAGAACATGTATGAACTGCAAAAAGGTAGTAGCGGCGGCACCTTGCTGGCCGGCATCGTGCAGCAACAACAGCAATTACTGGACGTGGTTAAACGCCAGCAAGAGATGCTTCGTCTCACGGTGTGGGGTACAAAAAACCTGCAAGCTAGAGTTACGGCAGGCGGTTCCGGCGGTAATATGACCTGGCAGGAGTGGGAACAGAAAGTGCGCTACCTGGAAGCCAACATTTCTCAGTCTCTTGAGCAAGCGCAGATTCAGCAAGAAAAGAACATGTACGAACTGCAAAAGCTGAATAGC.

[0046] In this invention, the nucleic acid may contain only the sequence shown in SEQ ID NO:2 or SEQ ID NO:4, or it may be a DNA molecule with the sequence shown in SEQ ID NO:2 or SEQ ID NO:4 as the coding region and additional components added. These other components may include any components required in the art for artificially synthesizing gene sequences and expressing them via expression vectors, such as promoters, enhancers, Kozak sequences, etc.

[0047] A third aspect of the present invention provides a carrier containing the nucleic acid described in the second aspect.

[0048] In this invention, the vector can be obtained by inserting nucleic acid into an empty vector. The empty vector can be any vector known in the art, such as various commercially available plasmids, granules, bacteriophages and retroviruses, for example, plasmid pET30a can be selected.

[0049] A fourth aspect of the present invention provides a cell containing the carrier described in the third aspect.

[0050] In this invention, the vector can be transformed, transduced, or transfected into host cells using methods known in the art, such as chemical transformation using calcium chloride or high-voltage electroporation, to obtain the cells described in the fourth aspect. The host cells can be derived from hosts known in the art, including prokaryotic or eukaryotic hosts; for example, the host cells can be derived from *Escherichia coli*.

[0051] The fifth aspect of the present invention provides a method for preparing recombinant human immunodeficiency virus antigen, the method comprising: inducing expression in the cells described in the third aspect to obtain recombinant human immunodeficiency virus antigen.

[0052] In this invention, appropriate induction expression methods can be selected according to the type of recombinant vector and recombinant cells. For example, isopropyl-beta-D-thiogalactopyranoside (IPTG) can be added to induce expression.

[0053] The sixth aspect of this invention provides the use of the human immunodeficiency virus recombinant antigen described in the first aspect, or the nucleic acid described in the second aspect, or the vector described in the third aspect, or the cell described in the fourth aspect in the preparation of human immunodeficiency virus recombinant antigen and / or the preparation of human immunodeficiency virus detection products.

[0054] In some embodiments, the detection product for human immunodeficiency virus may be at least one of test strips, chips, and kits.

[0055] The test strip may include a test strip carrier and a recombinant human immunodeficiency virus antigen disposed on the test strip carrier, and it typically achieves detection based on the principle of immunochromatography. For example, detection based on the colloidal gold method can use visible colloidal gold particles as a marker, and the results can be directly observed. Detection can also be achieved using other immunochromatographic principles known in the art, and based on specific principles, the test strip carrier and the recombinant human immunodeficiency virus antigen disposed on the test strip carrier can be modified in accordance with the art.

[0056] A chip, also known as a detector array, may include a solid-phase carrier containing multiple detection units and recombinant human immunodeficiency virus (HIV) antigens immobilized within the detection units. Detection can be achieved based on enzyme-linked immunosorbent assay (ELISA) or other known detection principles. One or more detection units containing HIV recombinant antigens can be placed within the chip, and optionally, detection units for detecting other targets can be included. A sample to be tested can be added to the chip and brought into contact with each detection unit, thereby achieving simultaneous detection of HIV antibodies and other targets in the sample.

[0057] The kit can be a combination of human immunodeficiency virus recombinant antigen and other reagents required for detection. Other reagents required for detection can be calibrators, quality control products, reference products, buffer solutions, diluents, etc. Reagents can be selected according to known kit composition methods in the field.

[0058] The seventh aspect of the present invention provides a human immunodeficiency virus (HIV) detection product, the product comprising at least one of the following: the recombinant HIV antigen described in the first aspect, and / or the nucleic acid described in the second aspect, the vector described in the third aspect, and the recombinant HIV antigen prepared from cells described in the fourth aspect.

[0059] To further understand the present invention, specific embodiments are described below. The scope of protection of the present invention is not limited by the following embodiments.

[0060] Unless otherwise stated, the reagents used in the following examples are commercially available. Experimental methods in the following examples that do not specify specific conditions were performed according to standard methods and conditions, or as selected in the product instructions.

[0061] Example 1: Preparation of HIV-1 recombinant antigen The recombinant antigen in this embodiment has the structure shown in formula (1): NH2-FP-NHR-X1-CHR-MPER-L1-FP-NHR-X2-CHR-MPER-L2-FP-NHR-X3-CHR-MPER-COOH Formula (1).

[0062] Select the following region sequences: FP region sequence selection: mgaasmtltvqarqlls; NHR region sequence selection: Mgaasmtltvqarqllsgivqqqnnllraieaqqhllqltvwgikqlqarilaverylkdq; X1 region sequence selection (loop sequence of gp41 protein): qllgiwgcsgklicttavpwnas; CHR region sequence selection: Wsnksleqiwnhttwmewdreinnytslihslieesqnqqekneqelleldkwaslwnwf; L1 and L2 sequence selection: GSSGG; X2 and X3 sequence selection: GGSGG; Combined with formula (1), the following amino acid sequence is formed (SEQ ID NO:1): MTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHL LQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLELDKWAS.

[0063] Based on the above amino acid sequence, the corresponding nucleotide sequence (SEQ ID NO:2) was designed according to the codon preference of E. coli: ATGACCGTGCAAGCGCGCCAACTGCTGAGCGGCATCGTTCAACAGCAGAACAACCTGCTGCGTGCGATTGAAGCGCAGCAGCATTTACTGCAGCTGACGGTGTGGGGCATTAAACAACTGCAAGCGCGCATTCTGGCGGTGGAGCGCTATCTGAAAGATCAGCAGCTGCTGGGCATTTGGGGCTGCAGCGGCAAACTGATTTGCACCACCGCGGTGCCGTGGAACGCGAGCTGGAGCAACAAAAGCCTGGAACAGATTTGGAACAACATGACGTGGATGGAATGGGATCGTGAAATTAACAACTATACGAGCCTGATTCATAGCCTGATCGAAGAGAGCCAAAATCAGCAAGAAAAAAATGAGCAAGAATTACTGGAAGGCAGTAGCGGCGGTCAGTTATTAAGCGGTATCGTGCAGCAACAGAATAACTTACTGCGCGCCATTGAAGCGCAGCAACATCTGCTGCAACTGACCGTTTGGGGTATCAAACAGTTACAAGCGCGTATTCTGGCGGGCGGTAGTGGCGGCCATACCACGTGGATGGAGTGGGATCGTGAGATTAATAACTATACGAGCCTGATCCATAGTTTAATTGAAGAAAGTCAGAATCAGCAAGAAAAGAATGAACAAGAATTATTAGAAGGTAGCAGCGGCGGCCAACTGTTAAGCGGCATTGTGCAGCAGCAAAATAACCTGCTGCGCGCGATCGAAGCGCAGCAGCATCTGCTGCAGCTGACCGTTTGGGGTATTAAGCAGCTGCAAGCGCGCATTCTGGCGGGTGGCAGCGGTGGCCATACGACGTGGATGGAATGGGACCGCGAAATCAATAACTACACGAGCTTAATTCATAGCCTGATTGAAGAGAGTCAAAATCAGCAAGAAAAAAACGAGCAAGAACTGCTGGAGTTAGATAAATGGGCGAGC。

[0064] The target gene sequence, such as SEQ ID NO:2, was synthesized by a gene synthesis company. The target gene fragment was amplified by DNA polymerase chain reaction (PCR) using primers with the sequences GGAATTCCATATGACCGTGCAAGCGCGCCAAC and AATTCCTCGAGGCTCGCCCATTTATCTAACTCCAG.

[0065] The amplified target gene fragment and the target vector pET30a were digested with restriction endonucleases NdeI and XhoI. The digested fragments were then recovered using a gel extraction kit. The digested target gene and the pET30a vector (NdeI / XhoI) were then ligated using DNA ligase. The ligated product was then transformed into competent DH5 cells using a heat shock method. The transformed cells were plated on Kans resistant LB plates and incubated overnight at 37°C. After single colonies emerged, the size of the inserted target gene was verified by PCR. Correct clones were confirmed by Sanger sequencing to have a target gene sequence completely consistent with the design, and were designated pET30a-HIV-1.

[0066] After amplifying and culturing the expression plasmid pET30a-HIV-1 / DH5, the target plasmid was extracted using a commercial plasmid extraction kit. Then, the expression strain BL21(DE3) was transformed using the heat shock transformation method, plated on Kan selection plates, and single clones of BL21-A [pET30a-HIV-1 / BL21(DE3)] were picked.

[0067] The expression strain BL21-A was cultured in LB liquid medium containing 50 g / ml kanamycin and grown at 37°C until OD200. 600 When the concentration of the protein was 0.8, the temperature was lowered to 25°C, and 0.2 mM IPTG was added. Expression was induced overnight at this temperature. After induction, the bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes and then frozen at -20°C.

[0068] The frozen bacterial cells were resuspended at a concentration of 100 g / L in Buffer A: 50 mM PB, 300 mM NaCl, 10 mM imidazole, pH 8.0 solution. After thorough resuspending, the bacterial cells were homogenized three times using an 800 bar high-pressure homogenization method. Then, the cells were centrifuged at 12,000 rpm and 4 °C for 30 minutes using a high-speed refrigerated centrifuge to separate the supernatant and precipitate. The supernatant was purified using a Ni-NTA affinity chromatography column. After equilibrating the Ni-NTA packing material with 5 column volumes of Buffer A, the supernatant was centrifuged and then equilibrated again with 5 column volumes of Buffer A. Then, contaminating proteins were washed with 3 column volumes of Buffer B (50 mM PB, 300 mM NaCl, 100 mM imidazole, pH 8.0). Finally, the target protein was eluted with Buffer C (50 mM PB, 300 mM NaCl, 500 mM imidazole, pH 8.0). The eluted protein was then dialyzed against Buffer D (50 mM PB, 150 mM NaCl, pH 8.0) to obtain the target protein HIV-1. The purification process is illustrated in the figure below. Figure 1 As shown. The target protein was obtained by supernatant purification, indicating that the HIV1 recombinant antigen in this embodiment is soluble and expressible.

[0069] Example 2: Preparation of HIV2 recombinant antigen The recombinant antigen in this embodiment has the structure shown in formula (1): NH2-FP-NHR-X1-CHR-MPER-L1-FP-NHR-X2-CHR-MPER-L2-FP-NHR-X3-CHR-MPER-COOH Formula (1).

[0070] Select the following region sequences: FP region sequence selection: LATAGSAMGAS; NHR region sequence selection: LTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKD; X2 region sequence selection (loop sequence of gp36 protein): QAQLNSWGCAFRQVCHTSVPWVNDTLT; CHR region sequence selection: PDWNNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQK; MPER region sequence selection: None L1 and L2 sequence selection: GSSGG; X1 and X3 sequence selection: GGSGG; Combined with formula (1), the following amino acid sequence is formed (SEQ ID NO:3): MLATAGSAMGAASLTLSAQSRTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAGGSGGNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQKGSSGGTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAIEKYLKDQA QLNSWGCAFRQVCHTSVPWVNDTLTPDWNNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQKGSSGGTLLAGIVQQQQQLLDVVKRQQEMLRLTVWGTKNLQARVTAGGSGGNMTWQEWEQKVRYLEANISQSLEQAQIQQEKNMYELQKLNS. Based on the above amino acid sequence, the corresponding nucleotide sequence (SEQ ID NO:4) was designed according to the codon preference of E. coli: ATGCTAGCGACAGCTGGGTCAGCAATGGGAGCGGCAAGCCTGACCCTGAGCGCACAGTCTCGTACCCTGCTGGCGGGCATCGTGCAGCAGCAACAACAGCTGCTGGACGTTGTTAAACGTCAGCAGGAGATGTTACGTCTGACCGTTTGGGGCACCAAGAACCTGCAGGCGCGTGTAACTGCGGGTGGTTCGGGTGGCAACATGACCTGGCAAGAGTGGGAGCAGAAGGTGCGTTACCTGGAGGCGAATATCAGCCAGAGCTTGGAGCAGGCGCAGATTCAGCAAGAGAAGAACATGTACGAACTGCAGAAGGGTTCCAGCGGTGGCACCCTGTTGGCCGGTATCGTCCAGCAACAACAACAATTGTTGGACGTCGTGAAGCGCCAGCAAGAAATGCTGCGTTTGACGGTCTGGGGTACGAAAAACCTGCAGGCTCGCGTGACCGCGATTGAAAAATATTTAAAAGATCAGGCTCAGTTGAACAGCTGGGGGTGCGCATTCCGCCAGGTTTGTCACACCAGCGTTCCGTGGGTTAATGATACCCTGACTCCGGATTGGAATAATATGACCTGGCAGGAGTGGGAACAAAAGGTACGTTATTTGGAAGCGAACATCTCCCAGTCACTGGAACAGGCGCAAATCCAACAAGAGAAGAACATGTATGAACTGCAAAAAGGTAGTAGCGGCGGCACCTTGCTGGCCGGCATCGTGCAGCAACAACAGCAATTACTGGACGTGGTTAAACGCCAGCAAGAGATGCTTCGTCTCACGGTGTGGGGTACAAAAAACCTGCAAGCTAGAGTTACGGCAGGCGGTTCCGGCGGTAATATGACCTGGCAGGAGTGGGAACAGAAAGTGCGCTACCTGGAAGCCAACATTTCTCAGTCTCTTGAGCAAGCGCAGATTCAGCAAGAAAAGAACATGTACGAACTGCAAAAGCTGAATAGC。 The target gene sequence, such as SEQ ID NO:4, was synthesized by a gene synthesis company. Primers with the sequences GGAATTCCAT ATGCTAGCGACAGCTGGGTC and AATTCCTCGAGGCTATTCAGCTTTTGCAGTTC were used to amplify the target gene fragment via DNA polymerase chain reaction (PCR).

[0071] The amplified target gene fragment and the target vector pET30a were digested with restriction endonucleases NdeI and XhoI. The digested fragments were then recovered using a gel extraction kit. The digested target gene and the vector pET30a (NdeI / XhoI) were then ligated using DNA ligase. The ligated product was then transformed into competent DH5 cells using a heat shock method. After transformation, the cells were plated on Kans resistant LB plates and incubated overnight at 37°C. After single colonies grew, the size of the target gene inserted into the vector was verified by PCR. The correct clone was confirmed by Sanger sequencing to have a target gene sequence that was completely consistent with the design and was designated as pET30a-HIV-2.

[0072] Plasmid transformation and cell fermentation were performed in the same manner as in Example 1.

[0073] After the fermented cells were sonicated to lyse the bacteria, the supernatant and precipitate were separated by centrifugation at 10,000 rpm for 10 minutes. SDS-PAGE gel electrophoresis was used to determine the location of the target protein. The electrophoresis image for detecting the expression of the HIV2 recombinant antigen is shown below. Figure 2 As shown, Figure 2 In the text, M represents the protein marker, the control is blank BL21(DE3) bacteria, whole bacteria refers to the sample before centrifugation, and supernatant and precipitate refer to the supernatant and precipitate obtained after centrifugation.

[0074] according to Figure 2 It can be seen that HIV2 recombinant antigen was obtained in the supernatant, that is, HIV2 recombinant antigen is soluble and expressible.

[0075] Comparative Example The recombinant antigen in this comparative example has the structure shown in formula (2): NH2-FP-NHR-X1-CHR-MPER-COOH Equation (2) Based on the region sequence selection in Example 1, the following recombinant antigen sequence was obtained: MTVQARQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKDQQLLGIWGCSGKLICTTAVPWNASWSNKSLEQIWNNMTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE.

[0076] Based on the codon preference of E. coli, the above recombinant antigen sequence was designed with corresponding nucleotide sequences. Plasmids were prepared and transformed into E. coli in accordance with the method in Example 1. Expression was induced in E. coli, and it was found that the recombinant antigen was expressed as inclusion bodies and was insoluble.

[0077] In addition, the literature Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain also provides results on the expression of existing recombinant antigens as inclusion bodies, which are cited in their entirety here.

[0078] Example 3: Preparation and testing of HIV colloidal gold test strips The HIV-1 protein prepared and purified in Example 1 was diluted to 0.7 mg / ml with coating solution, and then coated with 0.8 l / cm nitrocellulose membrane and dried for later use.

[0079] Preparation of colloidal gold labels: The preparation of colloidal gold is publicly known in the field, and a simplified process is as follows: Take 100 ml of 0.01% chloroauric acid solution, heat to boiling, then quickly add 1.6 ml of 1% trisodium citrate solution, continue stirring and boiling for 15 minutes, allow to cool naturally to room temperature, and add purified water to 100 ml to obtain colloidal gold solution. Adjust the pH of the prepared colloidal gold solution to 6.7 with 0.2M potassium carbonate solution, add HI-labeled antigen to a final concentration of 10 g / ml, stir magnetically for 15 minutes, and then add 1% BSA blocking colloidal gold. Centrifuge the labeled gold-labeled antigen at low speed, discard the supernatant, and obtain the concentrated gold-labeled antigen. Dilute the concentrated gold-labeled antigen to 20% of its original volume using gold-labeled diluent; evenly coat the gold-labeled antigen onto a glass fiber membrane, dry at 37℃ for 4 hours, and prepare an HIV gold-labeled antigen binding pad.

[0080] The HIV antigen-coated nitrocellulose membrane, HIV gold-labeled antigen-binding pad, sample pad, and absorbent paper are assembled into the HIV antibody test kit casing.

[0081] Using commercially available common HIV antigen (obtained through denaturation and renaturation) as a control, positive control P1 (high concentration control, colorimetric intensity > 5) and P2 (low concentration control, colorimetric intensity > 3) were compared. The HIV detection reagent obtained from the HIV-1 protein prepared and purified in Example 1 showed comparable sensitivity and specificity to the existing control material, at L7+ and L4+ (colorimetric card interpretation results), respectively. Furthermore, no false positives were found in 138 clinical negative samples prepared and purified in Example 1, indicating good specificity.

[0082] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A recombinant antigen of human immunodeficiency virus, characterized in that, The antigen has the structure shown in formula (1): NH2-FP-NHR-X1-CHR-MPER-L1-FP-NHR-X2-CHR-MPER-L2-FP-NHR-X3-CHR-MPER-COOH Formula (1); Among them, one or two of X1, X2 and X3 are each independently a loop sequence, and the rest are each independently a linking peptide; Among them, L1 and L2 are each independently a linker peptide; Among them, FP and MPER can be either none, a partial sequence, or a complete sequence, respectively.

2. The human immunodeficiency virus recombinant antigen according to claim 1, wherein, The loop sequence includes the loop sequence of the gp41 or gp36 protein; And / or, the sequence length of the linker peptide is 3-10 amino acids, preferably 5-7 amino acids; And / or, the linker peptide includes GGSSG and / or GGGGS.

3. The human immunodeficiency virus recombinant antigen according to claim 1 or 2, wherein, The antigen has the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:

3.

4. A nucleic acid encoding the recombinant antigen of human immunodeficiency virus as described in any one of claims 1-3.

5. The nucleic acid according to claim 4, characterized in that, The nucleic acid has the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:

4.

6. A carrier, characterized in that, The vector contains the nucleic acid as described in claim 4 or 5.

7. A cell, characterized in that, The cell contains the carrier as described in claim 6.

8. A method for preparing recombinant antigen of human immunodeficiency virus, characterized in that, The method includes: inducing expression in the cells of claim 7 to obtain recombinant human immunodeficiency virus antigen.

9. The use of the human immunodeficiency virus recombinant antigen according to any one of claims 1-3, or the nucleic acid according to claim 4 or 5, or the vector according to claim 6, or the cell according to claim 7 in the preparation of human immunodeficiency virus recombinant antigen and / or the preparation of human immunodeficiency virus detection products.

10. A detection product for human immunodeficiency virus, characterized in that, The product contains human immunodeficiency virus recombinant antigen as described in any one of claims 1-3, and / or nucleic acid as described in claim 4 or 5, vector as described in claim 6, and human immunodeficiency virus recombinant antigen prepared from at least one of the cells described in claim 7.

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