Preparation method of HIV (Human Immunodeficiency Virus) envelope protein gp160

Abstract

The invention discloses a preparation method of an HIV (Human Immunodeficiency Virus) envelope protein gp160. The preparation method comprises an effective interception and selection mode of amino acid sequences of envelope proteins gp160-I and gp160-II of HIV-I and HIV-II, an optimized nucleotide sequence, an expression plasmid construction method and a purification and preservation method. The gp160-I protein and the gp160-II protein are expressed through mammalian cells, the protein expression efficiency and the protein folding modification accuracy are greatly improved by adding the artificial signal peptide and the JSD promoter, and the gp160-I protein and the gp160-II protein have high biological activity and thermal stability and have the advantages that fermentation is easy to amplify, separation and purification are easy, and the coating use amount is low.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing HIV envelope protein gp160. Background Technology

[0002] gp160 plays a crucial role in HIV infection, serving as a key precursor to the viral envelope glycoprotein and directly driving the core process of HIV invasion of host immune cells. Synthesized within host cells, gp160 is cleaved by proteases into two functional units: gp120 and gp41. gp120 (the extracellular subunit) is primarily responsible for the precise recognition and binding of CD4 receptors and co-receptors (CCR5 / CXCR4) on the host cell surface, while gp41 (the transmembrane subunit) drives the fusion of the viral envelope with the host cell membrane, releasing viral genetic material. The correct synthesis, cleavage, and assembly of gp160, as well as the function of its derivatives gp120 / gp41, directly determine whether HIV can successfully invade host cells, representing one of the most critical steps in the viral life cycle.

[0003] gp160 (and its lysis products gp120 and gp41) are key proteins on the viral envelope and crucial for viral entry into cells. They are highly immunogenic, and the antibody response produced by the human body against them is early, strong, and very long-lasting. When a person is infected with HIV, the immune system produces antibodies against various viral proteins. Due to the extremely high immunogenicity of gp160, a large proportion of the antibodies produced by HIV-infected individuals target the envelope protein precursor gp160, which contains two functional units: gp120 and gp41.

[0004] gp160 is highly specific; these proteins are unique to HIV. HIV-infected individuals have a large number of antibodies against gp160 or its degradation units gp120 and gp41, but antibodies against them are almost non-existent in uninfected individuals.

[0005] While HIV-1 and HIV-2 gp160 cells share structural and functional similarities, they also exhibit significant differences. The amino acid sequence homology between the two is relatively low, ranging from approximately 40% to 60%, with the exact value varying depending on the region and strain being compared. This implies certain structural differences. The cleavage products of HIV-1 gp160 are gp120 and gp41, while those of HIV-2 are gp105 (or sometimes still called gp120, but in different sizes) and gp36 (sometimes still called gp41). gp105 is slightly smaller than HIV-1 gp120, while gp36 is slightly smaller than HIV-1 gp41 (although the gp160 precursor is slightly larger overall). Despite these differences, both HIV-1 and HIV-2 gp160 cells and their cleavage products possess strong immunogenicity and specificity. However, due to significant sequence and structural differences, antibodies generated against HIV-I gp120 / gp41 typically have weak or no neutralizing ability against HIV-II, and vice versa.

[0006] Because the HIV gp160 envelope protein is highly immunogenic and specific, it is clinically used to indirectly reflect whether an individual is infected with HIV by detecting antibodies against gp160 / gp120 / gp41. For example, third-generation HIV testing detects only antibodies, with a window period of approximately 3-12 weeks post-infection. Fourth-generation testing detects both HIV antibodies (based on the same principle, but with antigens including gp160 / gp41, etc.) and p24 antigen, shortening the window period to 2-6 weeks. The basic principle of using gp160 as a capture antigen for HIV detection is as follows: First, the gp160 protein, expressed using recombinant technology, is immobilized on a solid-phase detection device as a capture antigen. When a blood or serum sample is added, if the sample comes from an HIV-infected individual, the specific antibodies against gp160, gp120, or gp41 present in the sample will be captured and bound by the solid-phase antigen. Subsequently, an enzyme-labeled or colloidal gold-labeled secondary antibody is added. This secondary antibody specifically recognizes and binds to the human antibody, forming a complex of "solid-phase antigen-patient antibody-labeled secondary antibody." After washing to remove unbound components, the signal is amplified and visualized through appropriate colorimetric reactions or direct colorimetric development. Finally, the presence and intensity of the colorimetric reaction determine the presence of HIV-specific antibodies in the sample, thus achieving an indirect diagnosis of HIV infection.

[0007] In in vitro diagnostic kits for HIV testing, the core ingredient is the gp160 protein. Some bioactive ingredient companies typically obtain high-purity gp160 protein with a structure close to its natural form through recombinant expression. The main technologies involve three levels: first, the selection of the gene source (HIV-I and HIV-II gp160 genes); second, the selection of the expression system; and finally, the purification process (requiring high-purity antigen). Prokaryotic cell (e.g., E. coli) expression of HIV gp160 protein has several drawbacks, including glycosylation deficiency, difficulty in inclusion body refolding, and complex purification procedures, which prevent it from meeting the structural and functional complexity requirements of the protein. Eukaryotic cells can better preserve the native conformation and glycosylation modifications, but this results in lower expression rates and higher expression costs. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing HIV envelope protein gp160, including the selection of effective gp160 gene fragments, the design of artificial signal peptides, the construction of plasmid promoters, protein recombinant expression, and purification and preservation methods. The obtained gp160 has excellent structure and function, and is stable under preservation conditions, making it a reliable diagnostic raw material.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for preparing HIV envelope protein gp160 includes the following steps:

[0011] S1. Link an artificial signal peptide to the N-terminus of the gp160 amino acid fragment to obtain the gp160 nucleotide.

[0012] S2. The eukaryotic expression gene KOZAK sequence was added to the 5' end of the gp160 nucleotide to artificially synthesize the gp160 nucleotide expression gene.

[0013] S3. The JSD promoter gene was artificially synthesized and cloned into the pCMV vector via homologous recombination to construct the transient expression vector pJND.

[0014] S4. The glutamine synthase gene was artificially synthesized, cloned into the transient expression vector pJND, and a stable expression vector pJNDSG was constructed.

[0015] S5. The gp160 nucleotide expression gene was cloned into the stable expression vector pJNDSG to construct the stable recombinant plasmid pJNDSG-gp160.

[0016] S6. After the stable recombinant plasmid pJNDSG-gp160 is stably expressed, it is then purified to obtain gp160 protein, which is then stored in storage buffer.

[0017] Preferably, in step S1, the gp160 amino acid fragment is either the gp160-Ⅰ amino acid fragment or the gp160-Ⅱ amino acid fragment extracted from the database, and the sequences of the gp160-Ⅰ amino acid fragment and the gp160-Ⅱ amino acid fragment are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0018] Preferably, in step S2 above, the sequences of the gp160-Ⅰ nucleotide expression gene and the gp160-Ⅱ nucleotide expression gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0019] Preferably, in step S1 above, the amino acid sequence of the artificial signal peptide is shown in SEQ ID NO.5, and the nucleotide sequence encoding the artificial signal peptide is shown in SEQ ID NO.6.

[0020] Preferably, in step S3 above, the JSD promoter gene sequence is shown in SEQ ID NO.7, and the nucleotide sequence of the transient expression vector pJND is shown in SEQ ID NO.8.

[0021] Preferably, in step S4 above, the restriction sites for cloning the glutamine synthase gene into the vector pJND are the Xba1 and Sap1 sites.

[0022] Preferably, in step S5 above, the restriction enzyme sites for cloning the gp160-I nucleotide expression gene and the gp160-II nucleotide expression gene into the pJNDSG expression vector are HindIII and EcoRⅤ sites, respectively.

[0023] Preferably, in step S6 above, the stable expression of the recombinant plasmid pJNDSG-gp160 includes the following steps:

[0024] (1) The stable recombinant plasmid was introduced into CHO-K1 cells by electroporation;

[0025] (2) Screening was performed using a medium containing MSX to obtain single-clone cell clusters;

[0026] (3) Detect the expression level of target protein in the culture supernatant of each monoclonal cell cluster and screen for high-expression stable strains, amplify and culture them step by step and establish cryopreservation;

[0027] (4) Resuscitate the high-expression stable strains and scale them up sequentially for suspension culture. Temperature regulation is carried out during the expression stage, and uridine is added during the culture process.

[0028] (5) After culturing for a period of time, collect the culture supernatant to obtain gp160 histone.

[0029] Preferably, in step S6 above, the purification method for gp160 protein uses nickel ion affinity chromatography and molecular sieve chromatography; the storage buffer for gp160 protein is 10mM Tris-NaCl buffer, which includes the following components: 100mM NaCl, 1mM DTT, 1M Urea, 10% Glycerol and 0.1% Proclin 300, and the storage temperature is 2-8℃.

[0030] The gp160 protein prepared by the above preparation method is either gp160-I protein or gp160-II protein.

[0031] Preferably, the aforementioned gp160 protein is used in the preparation of immunoassay reagents for diagnosing HIV infection.

[0032] The advantages of this invention are as follows: The gp160-I (HIV-I) and gp160-II (HIV-II) proteins provided by this invention are expressed in mammalian cells. The addition of an artificial signal peptide can guide the transport of newly synthesized proteins from the cytoplasm to specific subcellular locations such as the cell membrane, endoplasmic reticulum, or Golgi apparatus, participate in the correct folding of proteins, and promote the correct secretion of proteins into the extracellular space. The addition of the JSD promoter can efficiently promote the expression of the target protein gene. The expression efficiency and protein folding modification accuracy of the gp160 protein prepared by this invention are greatly improved, and it has high biological activity and thermal stability. It also has the characteristics of easy fermentation scale-up, simple separation and purification, and low coating amount. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the construction of the gp160-Ⅰ / Ⅱ transient expression plasmid of the present invention;

[0034] Figure 2 This is a schematic diagram of the construction of the gp160-Ⅰ / Ⅱ stable expression plasmid of the present invention;

[0035] Figure 3 This is the chromatogram of the gp160-Ⅰ / Ⅱ nickel column purification of the present invention;

[0036] Figure 4 This is the purified chromatogram of the gp160-Ⅰ / Ⅱ molecular sieve Superdex75 of the present invention;

[0037] Figure 5 This is a graph showing the activity of the gp160-I protein according to the present invention;

[0038] Figure 6 This is a graph showing the activity detection of the gp160-II protein of the present invention;

[0039] Figure 7This is a stability test diagram of the gp160-I protein of the present invention;

[0040] Figure 8 This is a stability assay diagram of the gp160-II protein of the present invention. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0042] A method for preparing HIV envelope protein gp160 includes the following steps:

[0043] S1. An artificial signal peptide is ligated to the N-terminus of the gp160 amino acid fragment to obtain the gp160 nucleotide; the gp160 amino acid fragment is either gp160-Ⅰ or gp160-Ⅱ amino acid fragment extracted from the database, and the sequences of the gp160-Ⅰ and gp160-Ⅱ amino acid fragments are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively; the amino acid sequence of the artificial signal peptide is shown in SEQ ID NO.5, and the nucleotide sequence encoding the artificial signal peptide is shown in SEQ ID NO.6;

[0044] S2. The eukaryotic expression element KOZAK sequence was added to the 5' end of the gp160 nucleotide to artificially synthesize the gp160 nucleotide expression gene; the sequences of the gp160-Ⅰ nucleotide expression gene and the gp160-Ⅱ nucleotide expression gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively;

[0045] S3. The JSD promoter gene was artificially synthesized. The JSD promoter gene sequence is shown in SEQ ID NO.7, and the nucleotide sequence of the transient expression vector pJND is shown in SEQ ID NO.8. The transient expression vector pJND was constructed by cloning into the pCMV vector through homologous recombination.

[0046] S4. The glutamine synthase gene was artificially synthesized and cloned into the transient expression vector pJND through the restriction sites Xba1 and Sap1 to construct the stable expression vector pJNDSG.

[0047] S5. The gp160 nucleotide expression gene was cloned into the stable expression vector pJNDSG to construct the stable recombinant plasmid pJNDSG-gp160, which was then transformed into conventional Ecoli K-12 prokaryotic host cells. The restriction enzyme sites of the gp160-I nucleotide expression gene and the gp160-II nucleotide expression gene cloned into the pJNDSG expression vector were HindIII and EcoRⅤ sites, respectively.

[0048] S6. The stable recombinant plasmid pJNDSG-gp160 was purified by nickel affinity chromatography and molecular sieve chromatography to obtain gp160 protein, which was then stored in a storage buffer. The storage buffer for gp160 protein was a 10mM Tris-NaCl buffer, which consisted of the following components: 100mM NaCl, 1mM DTT, 1M Urea, 10% Glycerol, and 0.1% Proclin 300. The storage temperature was 2-8℃.

[0049] gp160-Ⅰ and gp160-Ⅱ were obtained from the NCBI database (Sequence ID: ADT65143.1 and Sequence ID: AID59535.1), respectively. Homology comparison and dominant antigenic epitope prediction were performed using the website. The effective amino acid fragments of gp160-Ⅰ and gp160-Ⅱ are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Nucleotide optimization was performed on gp160-Ⅰ and gp160-Ⅱ to improve their efficient expression in mammalian cells. The optimized nucleotide sequences of gp160-Ⅰ and gp160-Ⅱ are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0050] In eukaryotic expression systems, a signal peptide is a specific amino acid sequence (typically 15-30 residues) at the N-terminus of a protein. Its core function is to guide newly synthesized proteins into the secretory pathway, ensuring their correct folding, modification, and localization, and finally being cleaved by a signal peptidase. To better promote the expression, modification, and secretion of gp160 in mammalian cells, an optimized artificial signal peptide was linked to the N-terminus of gp160 amino acids. The amino acid sequence of the artificial signal peptide and the nucleotide sequence encoding it are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0051] The selected expression plasmid was the pCMV vector. To improve expression efficiency, the pCMV plasmid was modified by replacing the original promoter with the JSD promoter, constructing the expression vector pJND. The promoter sequence is shown in SEQ ID NO.7, and the expression vector pJND sequence is shown in SEQ ID NO.8. The optimized gp160-Ⅰ and gp160-Ⅱ genes containing the signal peptide were cloned into the pJND expression vector through HindⅢ and EcoRⅤ sites, respectively, to construct the pJND-gp160-Ⅰ / p160-Ⅱ recombinant expression plasmid, as shown below. Figure 1 As shown.

[0052] To construct a stable gp160 expression cell line, the glutamine synthase gene was cloned into the expression plasmids pJND-gp160-Ⅰ and pJND-gp160-Ⅱ, thus constructing stable expression plasmids pJNDSG-gp160-Ⅰ and pJNDSG-p160-Ⅱ. MSX pressure selection was used to obtain stable gp160-Ⅰ / Ⅱ expression cell lines. Figure 2 As shown in the figure. In this invention, by screening under pressure at a final concentration of 50 μM MSX, plasmids pJNDSG-gp160-Ⅰ and pJNDSG-p160-Ⅱ containing the glutamine synthase gene can be integrated into CHO, thus obtaining a cell line that stably expresses gp160.

[0053] Example 1, Construction of gp160-Ⅰ and gp160-Ⅱ expression plasmids, including the following steps:

[0054] (1) Based on the extracted effective gp160-I and gp160-II amino acid fragments (SEQ ID NO.1 and SEQ ID NO.2), artificial signal peptide amino acid sequences were linked to the N-terminus of the two fragments, and gp160-I and gp160-II nucleotides were optimized to obtain gp160-I and gp160-II nucleotides. The eukaryotic expression element KOZAK sequence was added to the 5' end of the nucleotides, and the gp160-I and gp160-II nucleotide expression genes (SEQ ID NO.3 and SEQ ID NO.4) were obtained through artificial synthesis.

[0055] (2) The JSD promoter gene sequence was artificially synthesized and cloned into the pCMV vector by homologous recombination to construct the transient expression vector pJND;

[0056] (3) The glutamine synthetase gene sequence was artificially synthesized and cloned into the pJND vector through the Xba1 and Sap1 restriction sites to construct a stable expression vector pJNDSG;

[0057] (4) The gp160-I and gp160-II nucleotide expression genes were cloned into the pJND expression vector through HindIII and EcoRⅤ sites to construct transient recombinant expression plasmids pJND-gp160-I and pJND-gp160-II. The constructed transient recombinant plasmids were transformed into conventional EcoliK-12 prokaryotic host cells. The plasmids were amplified by EcoliK-12, and 1 mg of endotoxin-free plasmids were extracted.

[0058] (5) The gp160-Ⅰ and gp160-Ⅱ nucleotide expression genes were cloned into the pJNDSG expression vector through HindⅢ and EcoRⅤ sites to construct stable reexpression plasmids pJNDSG-gp160-Ⅰ and pJNDSG-gp160-Ⅱ. The constructed stable recombinant plasmids were transformed into conventional EcoliK-12 prokaryotic host cells. The plasmids were amplified by EcoliK-12, and 500 μg of endotoxin-free plasmids were extracted.

[0059] Example 2, transient expression of gp160-Ⅰ and gp160-Ⅱ, includes the following steps:

[0060] (1) Transiently expressing HEK293 cells were cultured in HEK medium until the cell density increased to 3 × 10⁻⁶. 6 Cells / mL, prepare 1L of culture using a 2.5L sterile culture flask, and increase the density to 4×10⁻⁶. 6 CHO cells per mL;

[0061] (2) Measure 2 mL of sterile transfection reagent PEI (2 mg / mL) into 10 mL of PBS, mix thoroughly for 30 s using a vortex mixer, let stand for 10 min, and prepare 12 mL of PEI mixture.

[0062] (3) Add 1 mg of transient recombinant expression plasmid pJND-gp160-Ⅰ or 1 mg of pJND-gp160-Ⅱ to the PEI mixture, mix thoroughly for 30 s using a vortex mixer, and let stand for 10 min to prepare pJND-gp160-Ⅰ plasmid / PEI or pJND-gp160-Ⅱ plasmid / PEI mixture;

[0063] (4) Slowly add the pJND-gp160-Ⅰ plasmid / PEI or pJND-gp160-Ⅱ plasmid / PEI mixture to 1L of water until the density reaches 3×10⁻⁶. 6 Add the cells to the HEK293 cell culture solution at a ratio of 1 cell / mL while gently shaking the culture flask to ensure that the cells are thoroughly mixed with the PJND-gp160-Ⅰ plasmid / PEI or pJND-gp160-Ⅱ plasmid / PEI mixture.

[0064] (5) On the day after incubation, add uridine to a final concentration of 1 mM;

[0065] (6) On the third day of culture, the glucose concentration in the culture medium was measured, and the glucose concentration was replenished to 6 g / L.

[0066] (7) The cells were cultured at 35.5℃, 5% CO2, and 150 rpm / min with shaking. After 6 days of cell culture, the cells were centrifuged at 3500×g for 8 min and the expression supernatant of gp160-Ⅰ or gp160-Ⅱ was collected. The expression supernatant contained gp160-Ⅰ or gp160-Ⅱ recombinant protein, which was used for the next step of purification.

[0067] Example 3, stable expression of gp160-Ⅰ and gp160-Ⅱ, including the following steps:

[0068] (1) Inoculate one cell line of CHO-K1 into 50 mL of CHO medium containing 6 mM glutamine, and culture at 37°C, 5% CO2, and 150 rpm / min until the cell density reaches 3 × 10⁻⁶ cells / min. 6 cells / mL;

[0069] (2) Take 10 mL of the above cell culture and inoculate it into 100 mL of CHO medium containing 6 mM glutamine. Incubate at 37°C, 5% CO2, and 150 rpm / min until the cells are in the logarithmic growth phase, i.e., a cell density of 2 × 10⁻⁶. 6 Cells / mL, ensuring a viability of over 95%;

[0070] (3) Take 30 mL of the above cell culture medium, centrifuge at 1000 rpm for 5 min, remove the supernatant, add 5 mL of CHO medium to resuspend and wash once;

[0071] (4) Resuspend the cells in 800 μL of CHO medium, add 50 μg of endotoxin-free pJNDSG-gp160-Ⅰ or pJNDSG-gp160-Ⅱ stable reexpression plasmid, and prepare cell-plasmid mixture.

[0072] (5) Carefully pipette the above cell-plasmid mixture until it is evenly mixed, and quickly transfer it to a 0.4cm electroporation cup that has been chilled in an ice bath, so that the cells are kept in suspension as much as possible;

[0073] (6) Set the parameters of the bio-rad electroplasty device: exponential wave 300V, 800μF, 300Ω, 50ms, and perform electric shock;

[0074] (7) After the electric shock, carefully remove the white foam on the surface with a pipette, then add 200 μL of CHO culture medium and let stand for 10 min;

[0075] (8) Transfer the cells to 30 mL of CHO medium and place them in a cell culture incubator for 4 h of recovery. The recovery conditions are 37 °C and 5% CO2. Then transfer them to a shaking incubator for overnight culture. The culture conditions are 37 °C, 5% CO2 and 120 rpm / min.

[0076] (9) The next day, the cell viability was tested, and the viable cells were diluted to 1×10⁻⁶. 5 / mL, seeded into 96-well plates, 100μL per well, and placed in a cell culture incubator for static culture at 37℃ and 5% CO2;

[0077] (10) After 24 hours of plating, add 200 μL of CHO medium containing 50 μM MSX to each well for screening, and place in a 37°C, 5% CO2 incubator for static culture.

[0078] (11) Culture for 15-20 days, observe cell growth, and select viable, round monoclonal cell clusters;

[0079] (12) When the cell clusters grow to a certain size, take 100 μL of cell supernatant to detect protein expression. During this period, add 100 μL of CHO medium containing 50 μM MSX.

[0080] (13) Select high-expression single-cell clones and expand them into 24-well plates. After the cells have grown to confluence, transfer them to 6-well plates for further culture. After 4 days of expansion, expand them into 30 mL cell culture flasks.

[0081] (14) When the cell density in the cell culture flask reaches 2.0 × 10⁻⁶ 6 When the number of cells / mL is 1×10 7 Each frozen cell is a cell that stably expresses gp160-I or gp160-II.

[0082] (15) Take one vial of cryopreserved gp160-I or gp160-II stably expressing cells and seed them into 30 mL of CHO medium. The culture conditions are 37℃, 5% CO2, and 120 rpm / min. When the cell density reaches 2 × 10⁶ cells / min, the cells are cultured until the cell density increases to 2 × 10⁶ cells / min. 6 / mL;

[0083] (16) All 30 mL of cell culture medium was inoculated into 500 mL of CHO medium. The culture conditions were 37 °C, 5% CO2, and 120 rpm / min. The cell density was increased to 2 × 10⁻⁶ cells / min. 6 / mL;

[0084] (17) All of the contents were then inoculated into 1L CHO medium, and the expression conditions were 36℃, 5% CO2, and 120rpm / min.

[0085] (18) On the day after incubation, add uridine to a final concentration of 2mM;

[0086] (19) Check the glucose concentration in the culture medium every other day and replenish the glucose concentration to 6 g / L;

[0087] (20) After 14 days of cell culture, the cells were centrifuged at 3500×g for 8 min to collect the expression supernatant of gp160-I or gp160-II. The expression supernatant contained the recombinant protein of gp160-I or gp160-II for further purification.

[0088] Example 4: Purification of gp160-I and gp160-II proteins, including the following steps:

[0089] (1) Pack 10 mL of nickel ion packing into the chromatography column, equilibrate the chromatography column with 5-10 column volumes of ultrapure water, and then equilibrate the chromatography column with 100 mM PBS (100 mM NaCl, pH 7.4) at a flow rate of 5 mL / min, flushing to equilibrate 5-10 column volumes.

[0090] (2) Take 1L of gp160-Ⅰ or gp160-Ⅱ expression supernatant and filter it through a 0.45μM deep filter membrane, and add one-tenth of the volume of buffer 100mM PBS (0.1mM NaCl, pH 7.4);

[0091] (3) Load the processed expression supernatant slowly at a flow rate of 3 mL / min to ensure that the protein is fully bound;

[0092] (4) After loading the sample, equilibrate the chromatography column with 3 column volumes of equilibration buffer 100mM PBS (100mM NaCl, pH 7.4) at a flow rate of 5mL / min.

[0093] (5) Wash the chromatography column with 5 column volumes of washing buffer 100mM PBS (100mM NaCl, 20mM Imidazole, pH 7.4) at a flow rate of 5 mL / min;

[0094] (6) The protein was dissociated using 3 column volumes of dissociation buffer 100 mM PBS (100 mM NaCl, 200 mM Imidazole, pH 7.4), and the gp160 protein was collected (see the chromatogram of gp160-I / II nickel column purification). Figure 3 ), flow rate 3 mL / min;

[0095] (7) Prepare 100 mL of Superdex75 molecular sieve packing material and pack it into a 1 m long molecular sieve chromatography column;

[0096] (8) Equilibrate three column volumes with equilibration buffer 10mM Tris-NaCl (100mM NaCl, 1mM DTT, 1M Urea, 10% Glycerol);

[0097] (9) Load the gp160 protein obtained in step (6) onto a molecular superdex 75 sieve at a loading rate of 2 mL / min;

[0098] (10) Collect the main elution peak protein (gp160-I / II molecular sieve Superdex75 purification chromatogram, see below) Figure 4 This refers to the target protein of gp160;

[0099] (11) The storage buffer for gp160 target protein is 10mM Tris-NaCl (100mM NaCl, 1mM DTT, 1M MUrea, 10% Glycerol, 0.1% Proclin 300), and the storage temperature is 2-8℃.

[0100] Performance testing

[0101] An indirect capture ELISA method was used for detection. First, gp160-I or gp160-II protein was coated onto a 96-well ELISA plate, followed by blocking with BSA to exclude non-specific binding. Next, positive samples were added, allowing the specific anti-gp160 antibody to be captured by the coated antigen. Then, HRP-labeled secondary antibody M2811 was used for binding, and the signal was amplified by a colorimetric reaction. Finally, the absorbance at OD450 was measured, and the activity of the gp160 protein was determined based on the colorimetric intensity, as detailed below:

[0102] (1) Activity comparison: Experimental group: gp160-I or gp160-II protein expressed in eukaryotes according to this invention, 2 μg per well; Control group: gp160-I (CK) or gp160-II (CK) expressed in traditional prokaryotes, also 2 μg per well; Negative control group: no protein was coated. The higher the OD450 colorimetric value, the stronger the gp160 activity.

[0103] (2) Stability assessment: The gp160-I or gp160-II proteins expressed in this invention in the experimental group and the traditional prokaryotic expression gp160-I (CK) or gp160-II (CK) proteins in the control group were placed at 37℃ for assessment for 14 days. High-value positive samples were detected, and a negative control was set up. The higher the OD450 colorimetric value, the stronger the gp160 activity.

[0104] according to Figure 5 and Figure 6It can be seen that, compared with the conventionally expressed gp160-I (CK) or gp160-II (CK) at the same coating amount (2ug), the eukaryotically expressed gp160-I or gp160-II protein of the present invention has a stronger ability to capture specific anti-gp160 antibodies in positive samples, indicating that the gp160-I or gp160-II protein of the present invention has stronger activity.

[0105] according to Figure 7 and Figure 8 It can be seen that, after 14 days of testing at 37℃, the eukaryotic expression of gp160-Ⅰ or gp160-Ⅱ proteins of this invention showed a lower activity decrease compared to the conventionally expressed gp160-Ⅰ(CK) or gp160-Ⅱ(CK), indicating that the gp160-Ⅰ or gp160-Ⅱ proteins of this invention have stronger stability.

[0106] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing HIV envelope protein gp160, characterized in that, Includes the following steps: S1. Link an artificial signal peptide to the N-terminus of the gp160 amino acid fragment to obtain the gp160 nucleotide. S2. The eukaryotic expression gene KOZAK sequence was added to the 5' end of the gp160 nucleotide to artificially synthesize the gp160 nucleotide expression gene. S3. The JSD promoter gene was artificially synthesized and cloned into the pCMV vector via homologous recombination to construct the transient expression vector pJND. S4. The glutamine synthase gene was artificially synthesized, cloned into the transient expression vector pJND, and a stable expression vector pJNDSG was constructed. S5. The gp160 nucleotide expression gene was cloned into the stable expression vector pJNDSG to construct the stable recombinant plasmid pJNDSG-gp160. S6. After the stable recombinant plasmid pJNDSG-gp160 is stably expressed, it is then purified to obtain gp160 protein, which is then stored in storage buffer.

2. The method for preparing HIV envelope protein gp160 according to claim 1, characterized in that, In step S1, the gp160 amino acid fragment is either the gp160-Ⅰ amino acid fragment or the gp160-Ⅱ amino acid fragment extracted from the database. The sequences of the gp160-Ⅰ amino acid fragment and the gp160-Ⅱ amino acid fragment are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The amino acid sequence of the artificial signal peptide is shown in SEQ ID NO.5, and the nucleotide sequence encoding the artificial signal peptide is shown in SEQ ID NO.

6.

3. The method for preparing HIV envelope protein gp160 according to claim 2, characterized in that, In step S2, the sequences of the gp160-Ⅰ nucleotide expression gene and the gp160-Ⅱ nucleotide expression gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. The method for preparing HIV envelope protein gp160 according to claim 1, characterized in that, In step S3, the JSD promoter gene sequence is shown in SEQ ID NO.7, and the nucleotide sequence of the transient expression vector pJND is shown in SEQ ID NO.

8.

5. The method for preparing HIV envelope protein gp160 according to claim 1, characterized in that, In step S4, the glutamine synthase gene is cloned into the vector pJND, with the restriction sites being Xba1 and Sap1.

6. The method for preparing HIV envelope protein gp160 according to claim 3, characterized in that, In step S5, the gp160-I nucleotide expression gene and the gp160-II nucleotide expression gene are cloned into the pJNDSG expression vector at HindIII and EcoRⅤ sites, respectively.

7. The method for preparing HIV envelope protein gp160 according to claim 1, characterized in that, In step S6, the stable expression of the recombinant plasmid pJNDSG-gp160 includes the following steps: (1) The stable recombinant plasmid was introduced into CHO-K1 cells by electroporation; (2) Screening was performed using a medium containing MSX to obtain single-clone cell clusters; (3) Detect the expression level of target protein in the culture supernatant of each monoclonal cell cluster and screen for high-expression stable strains, amplify and culture them step by step and establish cryopreservation; (4) Resuscitate the high-expression stable strains and scale them up sequentially for suspension culture. Temperature regulation is carried out during the expression stage, and uridine is added during the culture process. (5) After culturing for a period of time, collect the culture supernatant to obtain gp160 histone.

8. The method for preparing HIV envelope protein gp160 according to claim 1, characterized in that, In step S6, the purification method for gp160 protein uses nickel ion affinity chromatography and molecular sieve chromatography; the storage buffer for gp160 protein is 10 mM Tris-NaCl buffer, which includes the following components: 100 mM NaCl, 1 mM DTT, 1 M Urea, 10% Glycerol and 0.1% Proclin 300, and the storage temperature is 2-8℃.

9. The gp160 protein prepared by the method according to any one of claims 1-8, characterized in that, The gp160 protein is either gp160-I or gp160-II.

10. The use of the gp160 protein of claim 9 in the preparation of an immunoassay reagent for diagnosing HIV infection.

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