A soluble high-activity HIV antigen fusion protein and a preparation method and application thereof

Abstract

The application discloses a soluble high-activity HIV antigen fusion protein and a preparation method and application thereof. The fusion protein comprises, in sequence from an N terminus to a C terminus, a truncated P64K protein fragment (SEQ ID NO: 1), a rigid connection peptide Linker (SEQ ID NO: 2), an HIV antigen fragment and a 6xHIS fusion label; the truncated P64K fragment corresponds to the C terminal 217-594 of the P64K protein, and retains a core solubilizing activity; the rigid Linker can effectively isolate the solubilizing label and the antigen, and avoid spatial steric hindrance. Through specific combination of the two, the soluble expression rate of the HIV antigen in E. coli reaches more than 85%, and the complete folding of an antigen epitope is guaranteed. The application further discloses an expression vector containing a T7 promoter and a preparation method. The fusion protein is high in purity and good in stability, can be widely applied to preparation of HIV in-vitro diagnostic reagents and antigen standard products, solves technical problems such as easy formation of inclusion bodies and low solubility of existing antigen expression, and has important clinical diagnostic value.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and immunoassay technology, and more specifically, to a soluble, highly active HIV antigen fusion protein, its preparation method, and its applications. Background Technology

[0002] Human immunodeficiency virus (HIV) infection is a major global public health problem. Despite continuous advancements in antiretroviral therapy, HIV infection remains incurable, and the risk of transmission persists. Early and accurate diagnosis of HIV infection is crucial for controlling the spread of the disease and reducing morbidity and mortality. HIV antigens, as the core raw material for diagnostic reagents, directly determine the sensitivity and specificity of these reagents, making them essential for early detection, intervention, and treatment of HIV infection. Therefore, developing high-quality, highly active HIV antigens is a core requirement for improving the performance of HIV diagnostic reagents and strengthening HIV prevention and control systems.

[0003] Currently, the core antigens used for HIV diagnosis mainly include gp41, gp120, and p24. Among them, prokaryotic expression systems have become the preferred system for HIV antigen preparation due to their low cost, high expression efficiency, and ease of large-scale production. Numerous cases of recombinant HIV antigen expression in various studies have been documented both domestically and internationally. For example, some literature uses GST and Trx tags to fusion express HIV-1 gp41 antigen (Elbahnasawy MA, et al. 2021; Penn-Nicholson, A., et al. 2007); Japanese patent application JP2009106289A discloses a method for expressing HIV gp120 and gp41 using a CKS fusion tag. However, due to the strong natural hydrophobicity and complex spatial conformation of HIV antigens, the solubilizing effects of the above methods are very limited. The proteins are mainly expressed in inclusion body form, making it difficult to form natural folds, which seriously affects antigen activity.

[0004] US Patent Application Publication No. US8426167B2 discloses a method for generating or enhancing the expression of fusion peptides. This technique utilizes FKBP-type molecular chaperones (including FkpA and SlyD) as fusion chaperones to bind flexible linker peptides. However, in this approach, the recombinant protein tends to form inclusion bodies after cytoplasmic expression. It requires denaturation and dissolution with high-concentration liquid salts (such as 6.0M guanidine hydrochloride), followed by complex in vitro refolding processes such as dialysis, rapid dilution, or on-column refolding to obtain soluble proteins. This denaturation and refolding process is not only lengthy but also costly. Furthermore, it is difficult to guarantee the conformational integrity of the antigenic epitope. Canadian patent application CA2214840A1 discloses an expression system containing a stable peptide derived from the first 47 amino acids of the N-terminus of the Neisseria meningitidis P64K antigen. This system utilizes this specific fragment to express the fusion protein at a high level in the form of inclusion bodies. Although this system improves the expression level, it still does not solve the technical problem of direct soluble accumulation of antigen in prokaryotic systems. Regarding the design of linker peptides, Chinese patent application CN101475642A discloses a scheme for co-expressing the fusion protein HT and HIV-1 gp41 in Escherichia coli. Although the solubility is improved, no linker peptide is set between the fusion protein and the target protein, which can easily lead to the fusion tag obscuring the antigenic epitope. Existing technologies often employ flexible linkers, such as the (GGGS)3GGG flexible linker used in Chinese patent application CN119137139A. However, due to the high degree of freedom in their molecular conformation, flexible linkers often cannot provide a stable steric barrier between the solubilizing tag and the target antigen, which can easily lead to steric hindrance, resulting in the masking or damage of key antigen epitopes and reduced immune activity.

[0005] P64K protein, an outer membrane protein with excellent solubilizing potential, has been used in existing technologies, but its full-length sequence is usually employed. Due to the large molecular weight of full-length P64K (approximately 60 kDa) and the presence of multiple strongly hydrophobic sites within its N-terminal 1-200 amino acid sequence, direct use as a fusion tag often reduces the overall expression level of the fusion protein and may even induce aggregation and precipitation, making it difficult to simultaneously improve solubility and maintain the biological function of the antigen. Based on this current technological status, there is an urgent need for an HIV fusion protein and its preparation method that can significantly improve the soluble expression rate of HIV antigens in prokaryotic systems, effectively avoid inclusion body refolding processes, and ensure complete exposure and high activity of antigenic epitopes. Summary of the Invention

[0006] One of the technical problems to be solved by the present invention is to provide a soluble, highly active HIV antigen fusion protein to solve the problems in the prior art where recombinant HIV antigens easily form inclusion bodies and fusion tags easily mask antigen epitopes, leading to reduced activity.

[0007] To overcome the shortcomings of the prior art, the present invention provides a soluble, highly active HIV antigen fusion protein, wherein the fusion protein comprises, from the N-terminus to the C-terminus, a truncated P64K protein fragment, a rigid linker peptide, an HIV antigen fragment, and a 6×HIS fusion tag.

[0008] The amino acid sequence of the truncated P64K protein fragment is shown in SEQ ID NO:1;

[0009] The amino acid sequence of the rigid linker peptide is shown in SEQ ID NO:2.

[0010] Compared with existing technologies, the soluble, highly active HIV antigen fusion protein of this invention has the following advantages: This invention precisely truncates the P64K solubilizing tag, discarding multiple strongly hydrophobic sites in its N-terminal amino acids 1 to 200, retaining only the C-terminal amino acid fragment from positions 217 to 594, which has highly efficient solubilizing and folding activities. This allows the solubilizing tag to maintain its efficient molecular chaperone function while reducing the overall metabolic burden and non-specific aggregation risk of the protein; further, through methods such as SEQ ID... The rigid helical structure (EAAAK)2 shown in NO:2 serves as a linker peptide. Its stable α-helical conformation in solution provides physical support, creating a defined spatial barrier between the truncated P64K fragment and the HIV antigen functional domain. This solves the problem of tag-antigen entanglement caused by the excessive conformational freedom of traditional flexible linkers. The specific spatial combination of the truncated tag and rigid isolation band in this invention achieves a high proportion of soluble accumulation of HIV antigen in the cytoplasm of *E. coli*, resulting in a soluble expression rate of over 85%. This avoids the high-concentration renaturation process required in existing technologies and ensures the full exposure and conformational integrity of key immunodominant epitopes of the HIV antigen, guaranteeing the efficient binding ability of the antigen to specific antibodies from a molecular-level perspective.

[0011] In one possible embodiment, the truncated P64K protein fragment has a molecular weight of 40.1 kDa; SEQ ID NO: 1 corresponds to amino acids 217 to 594 of the Neisseria meningitidis P64K protein, and the amino acid sequence of the Neisseria meningitidis P64K protein is shown in SEQ ID NO: 6.

[0012] In one possible implementation, the HIV antigen fragment is selected from one of the following: HIV-1 gp160, gp41 extracellular region, gp120 fragment, P24 antigen, HIV-2 gp140, gp36 extracellular region, and gp125 fragment; the amino acid sequence length of the HIV antigen fragment is 100-856 amino acids.

[0013] In one possible implementation, the amino acid sequence of the HIV antigen fragment is shown in SEQ ID NO:3.

[0014] In one possible implementation, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0015] In the above embodiments of the present invention, by precisely limiting the truncated P64K protein fragment to the amino acid range of positions 217 to 594, and within a specific molecular weight framework of 40.1 kDa, combining it with various HIV antigen fragments covering lengths of 100 to 856 amino acids, it is possible to ensure that the fusion tag provides optimal solubilization efficiency without generating additional expression load due to excessive molecular weight. Furthermore, the sequence selection and spatial arrangement shown in SEQ ID NO:3 and SEQ ID NO:4 achieve universal solubilization for HIV antigens of different lengths and physicochemical properties, ensuring that various antigenic epitopes can maintain a highly stable native conformation in complex aqueous environments, providing a definite structural basis for subsequent highly sensitive immunoassay.

[0016] This invention provides a gene sequence encoding the soluble, highly active HIV antigen fusion protein, as shown in SEQ ID NO:5. The gene sequence is a codon-optimized sequence tailored to E. coli codon preferences. Specifically, during the construction process, the original gene was artificially sequenced based on E. coli codon preferences. Synonymous substitutions were performed on rare codons in the heterologous sequence, and the base composition was adjusted to optimize the stability and translation rate of the mRNA in the host cell.

[0017] This invention provides a method for preparing the aforementioned soluble, highly active HIV antigen fusion protein, comprising the following steps:

[0018] Step S1: Construct a recombinant expression vector containing the gene sequence of the soluble, highly active HIV antigen fusion protein described above;

[0019] Step S2: Transform the recombinant expression vector into E. coli host cells, add an inducer to induce expression, and obtain bacterial cells;

[0020] Step S3: The bacterial cells are broken down and purified by affinity chromatography to obtain the soluble, highly active HIV antigen fusion protein.

[0021] The method provided by this invention lays the production foundation by constructing an expression vector containing the target gene in step S1; in step S2, the vector is transferred into E. coli and the expression process is started using an inducer, thereby obtaining bacterial cells containing recombinant protein; in step S3, the protein is separated and purified by a disruption and chromatography process, and finally a biologically active soluble fusion protein is obtained.

[0022] In one possible implementation, in step S1, the recombinant expression vector includes a plasmid with a pET series vector as its backbone, and the recombinant expression vector contains a T7 promoter and an resistance gene; in step S2, the inducer is IPTG, and the induction temperature is one of 16℃, 20℃, and 25℃; in step S3, the affinity chromatography is Ni-NTA affinity chromatography, and the soluble, highly active HIV antigen fusion protein is directly extracted from the cytoplasmic supernatant of the bacterial cells in a soluble form without undergoing renaturation treatment.

[0023] In the above embodiments, by using the pET series vector backbone in conjunction with the T7 promoter and resistance gene to construct expression plasmids, high-pressure transcriptional regulation and screening pressure maintenance of the target protein were achieved. Furthermore, by inducing expression with IPTG at low temperatures of 16℃, 20℃, or 25℃, the folding rate of the nascent polypeptide chain was slowed down, promoting the correct accumulation of the protein in the cytoplasm in its native conformation. In addition, the target product was directly extracted from the cytoplasmic supernatant using Ni-NTA affinity chromatography technology, without the need for renaturation treatment with guanidine hydrochloride or urea during the reaction, which simplified the production process while ensuring the structural integrity of the antigen epitope.

[0024] Another technical problem to be solved by the present invention is to provide the application of the soluble, highly active HIV antigen fusion protein in the preparation of HIV in vitro diagnostic reagents or HIV antigen standards, so as to solve the problems of poor stability and limited detection sensitivity of existing HIV diagnostic raw materials.

[0025] To overcome the shortcomings of the prior art, the present invention provides the application of the soluble, highly active HIV antigen fusion protein in the preparation of HIV in vitro diagnostic reagents or HIV antigen standards.

[0026] In one possible implementation, the HIV in vitro diagnostic reagent is one of the following: HIV chemiluminescence assay reagent, HIV latex-enhanced immunoturbidimetric assay reagent, and HIV immunochromatographic assay reagent.

[0027] Given that the fusion protein prepared in this invention has not been damaged by any chemical denaturation during the folding process, it retains the complex three-dimensional conformation of the HIV antigen in its native state. When applied to detection systems such as magnetic microparticle chemiluminescence or immunochromatography, it can provide a reaction kinetic constant that is much higher than that of conventional folded proteins. By using this highly soluble fusion protein with fully open epitopes as a capture antigen or detection antigen, the selective capture capability of diagnostic systems for low-concentration samples can be improved, especially in the detection of HIV-1 M and O subtype antibodies, which exhibit excellent signal-to-noise ratio and clinical discrimination. This highly active antigen raw material reduces the requirements of the test kit for sample volume and reaction time, enabling the diagnostic reagent to maintain a high level of specificity and repeatability when facing the detection of different HIV subtype antibodies. This provides a highly robust biological raw material support for the subsequent clinical realization of early detection and early diagnosis of HIV infection. Attached Figure Description

[0028] Figure 1 This is a map of the pAK-HIV gp41 recombinant expression plasmid constructed in the embodiments of the present invention;

[0029] Figure 2 This is a schematic diagram of the molecular structure of the soluble, highly active HIV antigen fusion protein in this invention; the figure shows the spatial arrangement of the truncated P64K, rigid linker, HIV-1 gp41 fragment, and 6×HIS tag.

[0030] Figure 3 This is an SDS-PAGE electrophoresis image of the soluble, highly active HIV antigen fusion protein induced and expressed in Escherichia coli in this invention; where M is the protein molecular weight standard (Protein Marker), "precipitate" is the insoluble part after bacterial cell disruption, and "supernatant" is the soluble part after bacterial cell disruption.

[0031] Figure 4 This is an electrophoresis image (SDS-PAGE) of the soluble, highly active HIV antigen fusion protein purified by affinity chromatography in this invention; wherein, "fusion protein" is the purified target product band, and M is the protein molecular weight standard (Protein Marker). Detailed Implementation

[0032] First, those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0033] This invention provides a soluble, highly active HIV antigen fusion protein, which comprises, from the N-terminus to the C-terminus, a truncated P64K protein fragment, a rigid linker peptide, an HIV antigen fragment, and a 6×HIS fusion tag.

[0034] The amino acid sequence of the truncated P64K protein fragment is shown in SEQ ID NO:1;

[0035] The amino acid sequence of the rigid linker peptide is shown in SEQ ID NO:2.

[0036] As a preferred embodiment, the truncated P64K protein fragment has a molecular weight of 40.1 kDa; and SEQ ID NO: 1 corresponds to amino acids 217 to 594 of the Neisseria meningitidis P64K protein, the amino acid sequence of which is shown in SEQ ID NO: 6.

[0037] As a preferred embodiment, the HIV antigen fragment is selected from one of the following: HIV-1 gp160, gp41 extracellular region, gp120 fragment, P24 antigen, HIV-2 gp140, gp36 extracellular region, and gp125 fragment; the amino acid sequence length of the HIV antigen fragment is 100-856 amino acids.

[0038] As a preferred embodiment, the amino acid sequence of the HIV antigen fragment is shown in SEQ ID NO:3.

[0039] As a preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0040] This invention provides a gene sequence encoding a soluble, highly active HIV antigen fusion protein, as shown in SEQ ID NO:5; the gene sequence is a codon-optimized sequence tailored to the preferences of Escherichia coli.

[0041] This invention provides a method for preparing a soluble, highly active HIV antigen fusion protein, comprising the following steps:

[0042] Step S1: Construct a recombinant expression vector containing the gene sequence of a soluble, highly active HIV antigen fusion protein;

[0043] Step S2: Transform the recombinant expression vector into E. coli host cells, add an inducer to induce expression, and obtain bacterial cells;

[0044] Step S3: Disrupt the bacterial cells and purify them by affinity chromatography to obtain a soluble, highly active HIV antigen fusion protein.

[0045] As a preferred embodiment, in step S1, the recombinant expression vector includes a plasmid with a pET series vector as its backbone, and the recombinant expression vector contains a T7 promoter and an anti-resistance gene; in step S2, the inducing agent is IPTG, and the induction temperature is one of 16℃, 20℃, and 25℃; in step S3, the affinity chromatography is Ni-NTA affinity chromatography, and the soluble, highly active HIV antigen fusion protein is directly extracted from the cytoplasmic supernatant of the bacterial cells in a soluble form without undergoing renaturation treatment.

[0046] This invention provides the application of a soluble, highly active HIV antigen fusion protein in the preparation of HIV in vitro diagnostic reagents or HIV antigen standards.

[0047] As a preferred option, the HIV in vitro diagnostic reagent is one of the following: HIV chemiluminescence assay reagent, HIV latex-enhanced immunoturbidimetric assay reagent, and HIV immunochromatographic assay reagent.

[0048] This invention, based on protein engineering technology, truncates the P64K solubilizing tag, retaining only the C-terminal amino acid fragment from amino acids 217 to 594, which possesses core solubilizing and folding functions. This avoids the negative impact of the strongly hydrophobic N-terminal region of the full-length protein on recombinant expression, ensuring the solubility of the fusion protein in prokaryotic expression systems at the molecular structural level. Combined with a rigid linker peptide as shown in SEQ ID NO:2, its stable helical structure constructs an effective spatial barrier between the truncated P64K solubilizing tag and the HIV antigen functional domain, thus completely solving the steric hindrance problem caused by the excessive freedom of traditional flexible linkers. This ensures the full exposure of key immunodominant epitopes of the HIV antigen and accurate folding of its native conformation. Through this innovative combination of specific components, the soluble expression rate of recombinant HIV antigen in *E. coli* is increased to over 85%, and it can be directly purified by nickel column chromatography using cytoplasmic supernatant with high purity. The fusion protein provided by this invention not only solves the technical problems of HIV antigen prokaryotic expression being prone to inclusion body formation, complex processing, and easy damage to epitopes, but also shows significant application value in the development of diagnostic reagents such as HIV chemiluminescence detection, latex-enhanced immunoturbidimetry, and immunochromatography due to its excellent immunoreactivity and stability.

[0049] The following are embodiments incorporating specific data to further elaborate on the above-described technical solutions of the present invention:

[0050] Example 1: Soluble, highly active HIV antigen fusion protein and its preparation method

[0051] This embodiment provides a soluble, highly active HIV antigen fusion protein and its preparation method; the overall molecular configuration of the fusion protein is as follows: Figure 2As shown, the fusion protein comprises, from N-terminus to C-terminus, a truncated P64K protein fragment (the amino acid sequence of the P64K protein is shown in SEQ ID NO:6), a rigid linker peptide, an HIV-1 gp41 antigen fragment, and a 6×HIS fusion tag, arranged sequentially. The truncated P64K protein fragment is selected from amino acid positions 217 to 594 of the Neisseria meningitidis P64K protein, and its corresponding amino acid sequence is shown in SEQ ID NO:1. The rigid linker peptide is a derived peptide segment as shown in SEQ ID NO:2. The HIV antigen portion is the fragment shown in SEQ ID NO:3. The complete amino acid sequence of the fusion protein is shown in SEQ ID NO:4. The specific preparation method includes the following steps:

[0052] S1: Construction of the recombinant expression vector; Based on the protein sequence shown in SEQ ID NO:4, the nucleotide sequence was optimized by analyzing the codon preference of *E. coli*, thereby reducing the secondary structure of mRNA and improving translation efficiency, resulting in the gene sequence shown in SEQ ID NO:5; The whole gene synthesis was entrusted to Sangon Biotech Co., Ltd.; The synthesized target gene sequence completely contains the gene fragment encoding a truncated P64K, a rigid linker, the extracellular region of HIV-1 gp41, and a 6×His tag; Sangon Biotech Co., Ltd. used restriction endonucleases to directionally clone the synthesized target gene into the pET-28a expression vector, thereby constructing a recombinant expression vector named pAK-HIV gp41, the specific expression plasmid map of which is shown in the figure below. Figure 1 As shown;

[0053] S2: Transformation and Induction of Expression: The recombinant expression vector pAK-HIV gp41 constructed above was transformed into Escherichia coli BL21(DE3) competent cells, and then plated on LB solid medium plates containing 100 μg / ml kanamycin and incubated overnight at 37°C. Single colonies with good growth were picked from the plate using a sampling loop and inoculated into 30 ml of LB liquid medium and cultured with shaking at 37°C for 12-16 hours. The culture medium was used for PCR verification and sequencing identification to obtain a positive recombinant strain with a completely correct gene sequence. The identified positive recombinant strains were transferred to 10 mL of LB liquid medium containing 100 μg / mL kanamycin at the inoculum ratio. The culture was maintained at 37℃ and 200 rpm until the OD600 reached 0.6-0.8, serving as a qualified seed culture. The seed culture was then transferred to 1 L of fresh LB liquid medium at a 1:100 inoculum ratio, and the culture conditions were maintained at 37℃ and 200 rpm until the OD600 reached 0.6-0.8. At this point, IPTG was added to the culture system to a final concentration of 0.4 mM, and the culture environment was adjusted to 20℃ and 200 rpm. Expression was induced for 18 hours under low temperature conditions. After the induction phase, the bacterial cells were collected by centrifugation.

[0054] S3: Isolation and purification of the fusion protein; First, wash the collected bacterial cells twice with 10mM PBS buffer (pH 7.4) to remove surface impurities; Weigh 1g of the washed bacterial cells and resuspend them in 50ml of 20mM sodium acetate lysis buffer (pH 5.5), and add 1.5mmol / L deoxyribonuclease and 1.5mmol / L benzyl sulfonyl fluoride to the suspension; Lyse the bacterial cells using ultrasonication, setting the ultrasonic power to 35W and the total lysis time to 10 minutes; After lysis, centrifuge the mixture at 12000r / min for 10 minutes, and collect the supernatant and precipitate separately; Then, adjust the pH of the supernatant to 8.0 using sodium hydroxide solution, and filter it using a 0.45μm filter membrane, storing it at 4℃ for later use. Note that the filtered supernatant should not be left overnight.

[0055] The expression localization of the product was detected and verified using SDS-PAGE electrophoresis, referring to... Figure 3The induced expression electrophoresis diagram shown indicates that the target fusion protein is mainly enriched in the supernatant. The band grayscale analysis determined that its soluble expression rate reached 85%, and its molecular weight was approximately 59 kDa. This result is in complete agreement with the theoretical prediction. Next, the target protein in the supernatant was purified using nickel affinity chromatography. After packing the nickel packing material into the chromatography column, it was washed and equilibrated with 10 mM PB buffer at pH 8.0. The equilibration was determined to be complete when the fluctuation of the detector reading within 1 minute was less than 0.001, using a nucleic acid protein detector. The supernatant, after being adjusted to pH and filtered through a 0.45 μm filter membrane, was slowly loaded onto the column using a constant flow pump. After loading, impurities were first washed with 10 mM PB buffer at pH 8.0 containing 5 mM imidazole. Then, gradient elution was performed with 10 mM PBS buffer at pH 8.0 containing 50 mM, 100 mM, and 200 mM imidazole, respectively, and the target protein eluent was collected. After elution, the chromatography column was washed with buffer containing 500 mM imidazole and then regenerated using a 1 M high-concentration sodium hydroxide solution. The collected protein eluent was dialyzed into 10 mM PB buffer (pH 8.0). The dialysate was changed every 3 hours during dialysis, for a total of 4 changes. After dialysis, the obtained pAK-HIV gp41 fusion protein was stored at 4°C for later use. (Refer to...) Figure 4 The electrophoresis pattern after purification shown indicates that the purity of the final recombinant fusion protein is over 90%. The entire preparation process directly extracts the protein from the bacterial cytoplasm supernatant in a soluble form, completely avoiding the complex renaturation process, which can well meet the requirements of subsequent experiments and practical applications.

[0056] Example 2: Application and activity verification of soluble, highly active HIV antigen fusion protein in detection reagents

[0057] This embodiment provides the application of the soluble, highly active HIV antigen fusion protein prepared above in the preparation of HIV in vitro diagnostic reagents; specifically, the reactivity of the recombinant protein pAK-HIV gp41 in anti-HIV immunoassay was verified using magnetic microparticle chemiluminescence method; the test was conducted on the 2280 automated chemiluminescence analyzer of Meikang Biotechnology, using a dual antigen sandwich detection mode.

[0058] In constructing the detection system, the purified fusion protein was immobilized on the surface of carboxyl magnetic beads with a diameter of 0.8 μm as the capture antigen, and the same fusion protein was biotin-labeled as the detection antigen. The luminescent system used streptavidin labeled with acridinium ester. The test samples used were the national reference materials for human immunodeficiency virus antibodies provided by the National Institutes for Food and Drug Control. This reference material system includes negative reference materials (N1-N13), HIV-1 M positive reference materials (P1-P13), HIV-1 O positive reference materials (P15-P17), and HIV-2 positive reference materials (P18-P20).

[0059] The national reference samples of the above different groups were subjected to magnetic particle chemiluminescence detection and the response values ​​were recorded. The specific luminescence intensity RUL values ​​of each group of samples are recorded in Table 1 below: Table 1. RUL values ​​of HIV reference standards for pAK-HIV gp41 antigen determined by chemiluminescence immunoassay:

[0060] Further grouping and statistical parameter evaluation were performed on the raw data obtained from the above tests. The relevant statistical analysis results are summarized in Table 2 below: Table 2. Statistical results of HIV reference material determination using pAK-HIV gp41 antigen by chemiluminescence immunoassay:

[0061] Analysis of the detection data shown in Tables 1 and 2 reveals that the median Recovery Usage (RUL) for HIV-1 M subtype positive reference samples was 484,217, and the median RUL for HIV-1 O subtype positive reference samples was 543,011. Both values ​​are significantly higher than the median RUL of 3,321 for negative reference samples, demonstrating extremely clear clinical differentiation. In contrast, the median RUL for HIV-2 positive reference samples was 4,300, showing no significant difference from the negative reference samples. These experimental results demonstrate that the fusion protein prepared in this invention can specifically recognize HIV-1 M and O subtype antibodies and does not cross-react with HIV-2 antibodies.

[0062] A comparison of the preparation process and detection data from the above embodiments reveals that this invention, through the innovative combination of a specific truncated P64K fragment (amino acids 217-594) with a rigid linker, successfully achieved efficient accumulation of the pAK-HIV gp41 fusion protein in a prokaryotic system, with a soluble expression rate of approximately 85%. The rigid linker effectively isolates the P64K solubilizing tag from the HIV antigen functional domain, avoiding steric hindrance interference caused by spatial entanglement, thus ensuring the complete folding and full exposure of the antigen epitope. Validation data from the magnetic microparticle chemiluminescence assay further confirms that this fusion protein possesses excellent immunoreactivity and antigen specificity, making it a high-quality raw material suitable for the preparation of diagnostic reagents for HIV chemiluminescence, latex-enhanced immunoturbidimetry, or immunochromatography, providing reliable technical support for the early detection and diagnosis of HIV.

[0063] In the description of this invention, the references to "one embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0064] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A soluble, highly active HIV antigen fusion protein, characterized in that, The fusion protein, from N-terminus to C-terminus, comprises: a truncated P64K protein fragment, a rigid linker peptide, an HIV antigen fragment, and a 6×HIS fusion tag. The amino acid sequence of the truncated P64K protein fragment is shown in SEQ ID NO:1, and the molecular weight is 40.1 kDa; SEQ ID NO:1 corresponds to amino acids 217 to 594 of the Neisseria meningitidis P64K protein, and the amino acid sequence of the Neisseria meningitidis P64K protein is shown in SEQ ID NO:

6. The amino acid sequence of the rigid linker peptide is shown in SEQ ID NO:2; The HIV antigen fragment is an extracellular antigen fragment of HIV-1 gp41, and its amino acid sequence is shown in SEQ ID NO:

3.

2. The soluble, highly active HIV antigen fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

3. A gene encoding the soluble, highly active HIV antigen fusion protein of claim 2, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO:5; the nucleotide sequence is a codon-optimized sequence tailored to the preferences of Escherichia coli.

4. A method for preparing a soluble, highly active HIV antigen fusion protein, characterized in that, Includes the following steps: Step S1: Construct a recombinant expression vector containing the gene described in claim 3; Step S2: Transform the recombinant expression vector into E. coli host cells, add an inducer to induce expression, and obtain bacterial cells; Step S3: The bacterial cells are broken down and purified by affinity chromatography to obtain the soluble, highly active HIV antigen fusion protein.

5. The preparation method according to claim 4, characterized in that, In step S1, the recombinant expression vector includes a plasmid with a pET series vector as its backbone, and the recombinant expression vector contains a T7 promoter and an resistance gene. In step S2, the inducer is IPTG, and the induction temperature is one of 16℃, 20℃, and 25℃. In step S3, the affinity chromatography is Ni-NTA affinity chromatography, and the soluble, highly active HIV antigen fusion protein is extracted directly from the cytoplasmic supernatant of the bacterial cells in a soluble form without undergoing renaturation treatment.

6. The use of the soluble, highly active HIV antigen fusion protein according to claim 1 or 2 in the preparation of in vitro diagnostic reagents or HIV-1 antigen standards for detecting HIV-1 M and / or O subtype antibodies.

7. The application according to claim 6, characterized in that, The in vitro diagnostic reagent is one of the following: HIV-1 chemiluminescence immunoassay reagent, HIV-1 latex-enhanced immunoturbidimetric assay reagent, and HIV-1 immunochromatographic assay reagent.

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