HCV recombinant fusion antigen protein

Abstract

The invention discloses an HCV recombinant fusion antigen protein which comprises a Core antigen epitope, an NS3 antigen epitope region and an NS5A antigen epitope of HCV which are connected in series, the amino acid sequence of the Core antigen epitope is as shown in SEQ ID NO: 1, the amino acid sequence of the NS3 antigen epitope is as shown in SEQ ID NO: 2, and the amino acid sequence of the NS5A antigen epitope is as shown in SEQ ID NO: 3. The HCV recombinant fusion antigen protein obtained by the invention has relatively high detection rate and specificity.

Description

Technical Field

[0001] This invention relates to the field of HCV detection technology, and more particularly to an HCV recombinant fusion antigen protein. Background Technology

[0002] Hepatitis C (HCV) is a disease caused by infection with the hepatitis C virus (HCV), primarily transmitted through blood and bodily fluids. HCV is highly variable and exhibits pantropy for extrahepatic cells. Combined with its low titer in blood and weak immunogenicity, these biological characteristics make timely detection of the virus difficult, and the body's immune system struggles to effectively clear it. Chronic HCV infection has become a leading cause of cirrhosis and hepatocellular carcinoma (HCC), imposing a heavy socioeconomic burden on countries worldwide. Therefore, strengthening the prevention, screening, and treatment of HCV remains a crucial task in global public health.

[0003] HCV is a single-stranded positive-sense RNA virus with a genome approximately 9.5 kb in length, divided into three regions: the 5' untranslated region (5'UTR), the coding region (ORF), and the 3' untranslated region (3'UTR), arranged in the order 5'UTR-C-E1-E2-p7-NS2-NS3-NS4-NS5-3'UTR. The 5'UTR is a highly conserved region and serves as the initiation site for viral translation, playing a crucial role in HCV replication. The open reading frame (ORF) includes the structural protein regions C, E1, and E2, and the non-structural protein regions P7, NS2-NS5. The envelope regions (E1, E2) and the core region (C) encode the viral particle, while the non-structural protein regions play important roles in viral replication and viral protein synthesis.

[0004] Antibodies in the blood of HCV-infected patients mainly generate immune responses against the following antigenic epitopes: Core (2-120 aa), NS3 (1026-1457 aa), NS4 (1694-1735 aa), NS5A (1859-1931 aa), and NS5A (2212-2313 aa). Core and NS3 antigens show good complementarity in antigen reactivity, and their combined detection rate is about 99% on average, making them key targets for the development of HCV diagnostic reagents. Existing research has proposed a variety of innovative strategies. For example, CN113640519A proposes to design and prepare Core-NS3 fusion protein to improve HCV detection efficiency; CN105254724A advocates to construct a truncated HCV-NS3 antigen to optimize the detection process; CN102072957A innovatively uses specific HCV recombinant multiantigen components (i.e. gene fragments covering Core, NS3, NS4, and NS5) to label magnetic microparticles. In addition, CN107129539A proposes that Core antigen-NS5 fusion antigen has beneficial effects on improving the hydrophilicity, sensitivity, and stability of HCV protein.

[0005] Despite the existence of various HCV detection antigens in existing technologies, significant challenges remain in practical clinical applications. First, HCV exhibits extremely high variability, and combinations of single or dual antigens often fail to cover all genotypes, particularly in early-stage infections with antibodies against the NS5A region, leading to frequent false negatives. Second, traditional physical antigen mixing strategies suffer from the umbrella effect, where highly immunogenic antigens may spatially mask weakly immunogenic antigens, resulting in decreased detection sensitivity. Furthermore, the HCV core protein is naturally highly hydrophobic, readily forming non-specific aggregates in vitro. This not only reduces epitope exposure efficiency but also leads to excessively high background signals in the kit, causing false positives. Therefore, there is an urgent need in the field for a recombinant HCV antigen design scheme that can simultaneously address the challenges of broad spectrum, high sensitivity, and high solubility. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an HCV recombinant fusion antigen protein. This protein utilizes the Core, NS3, and NS5A gene sequences as dominant epitopes, analyzes their codon usage frequencies using bioinformatics software, and optimizes the codons of the three genes based on the codon preferences of *E. coli*. The resulting HCV recombinant fusion antigen protein exhibits high detection rate and specificity.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A recombinant HCV fusion antigen protein comprising a tandemly linked HCV Core antigenic epitope, an NS3 antigenic epitope region, and an NS5A antigenic epitope, wherein the amino acid sequence of the Core antigenic epitope is as shown in SEQ ID NO: 1, the amino acid sequence of the NS3 antigenic epitope is as shown in SEQ ID NO: 2, and the amino acid sequence of the NS5A antigenic epitope is as shown in SEQ ID NO: 3.

[0009] Preferably, the Core antigenic epitope, the NS3 antigenic epitope, and the NS5A antigenic epitope are linked by peptide linkers.

[0010] Preferably, the peptide linker is a flexible linker, and its amino acid sequence is shown in SEQ ID NO: 4.

[0011] Preferably, the recombinant protein has the amino acid sequence shown in SEQ ID NO: 5.

[0012] Preferably, the recombinant fusion antigen protein further comprises a purification tag at its N-terminus or C-terminus, wherein the purification tag is a histidine tag.

[0013] A nucleic acid molecule encoding an HCV recombinant fusion antigen protein.

[0014] Preferably, the nucleic acid molecule comprises the nucleotide sequences shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or fusion sequences of the same molecule linked by a linker sequence, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 9.

[0015] A recombinant expression vector containing nucleic acid molecules, wherein the recombinant expression vector is pET28a(+).

[0016] A host cell containing a recombinant expression vector, wherein the host cell is Escherichia coli BL21(DE3).

[0017] Application of HCV recombinant fusion antigen protein in the preparation of HCV antibody detection reagents.

[0018] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention uses a specific short flexible linker to tandem the core, NS3, and NS5A gene sequences. This specific length and flexibility of the linker successfully isolates each domain, preventing misfolding interference between domains, while giving each epitope sufficient spatial freedom to bind antibodies. Combined with thermodynamic stability codon optimization targeting the E. coli host system, this fusion protein achieves highly soluble expression in the supernatant, completely solving the cumbersome process problem of urea denaturation and renaturation in traditional HCV antigen preparation, and maximizing the preservation of native conformation epitopes. The resulting HCV recombinant fusion antigen protein has a high detection rate and specificity. Attached Figure Description

[0019] Figure 1 This is an electrophoresis diagram of the present invention. Detailed Implementation

[0020] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0021] The present invention discloses a recombinant HCV fusion antigen protein comprising a tandemly linked HCV Core antigenic epitope, an NS3 antigenic epitope region, and an NS5A antigenic epitope, wherein the amino acid sequence of the Core antigenic epitope is as shown in SEQ ID NO: 1, the amino acid sequence of the NS3 antigenic epitope is as shown in SEQ ID NO: 2, and the amino acid sequence of the NS5A antigenic epitope is as shown in SEQ ID NO: 3.

[0022] The Core antigenic epitope, NS3 antigenic epitope, and NS5A antigenic epitope are linked by peptide linkers. These peptide linkers are flexible and their amino acid sequences are shown in SEQ ID NO: 4.

[0023] The selected core antigen epitope is 2-121aa, and the optimized nucleotide sequence is SEQ ID NO: 6. It retains the hydrophilic N-terminal Domain 1 (the main immunogenic region) and removes the C-terminal hydrophobic tail that leads to aggregation. This not only improves expression levels but, more importantly, prevents non-specific precipitation of the antigen during kit storage. The selected NS3 antigen epitope is 1435-1615aa. This region contains a helicase ATP-binding motif, with an intact domain, preserving the conformational epitope while avoiding hydrophobic polymerization of the full-length NS3, and covering the most enriched region for clinical antibody detection. The optimized nucleotide sequence is SEQ ID NO: 7. The selected NS5A antigen epitope is 2035-2124aa. This region is a natural low-complexity-acidic region, which is less prone to aggregation in E. coli. The optimized nucleotide sequence is SEQ ID NO: 8. The optimized core, NS3, and NS5A sequences were spliced ​​together, and the amino acid sequence is SEQ ID No: 5. The corresponding optimized nucleotide sequence is SEQ ID NO: 9.

[0024] Example 1: A method for constructing a recombinant expression vector, comprising the following specific steps:

[0025] (1) The optimized gene sequence encoding the HCV recombinant fusion antigen was obtained by chemical synthesis. The recombinant protein gene sequence was amplified by PCR and ligated into the Escherichia coli expression vector pET28a(+) by T4 ligase overnight at 4 ℃.

[0026] (2) The recombinant plasmid was transformed into competent E. coli BL21(DE3) cells and plated on LB agar plates containing 50 μg / mL Kan, and cultured at 37 °C for 16 h. Single colonies were picked, and positive strains that were identified by bacterial PCR and double enzyme digestion were selected for preservation.

[0027] Example 2: Induction of recombinant protein expression, including the following specific steps:

[0028] (1) Positive clones were picked and inoculated into LB medium containing 50 μg / mL Kan and cultured with shaking at 37 ℃. After the OD600 reached 0.6-0.8, 1.0 mM IPTG was added and cultured at 37 ℃ for 4 h. The cells were collected by centrifugation and the expression of recombinant proteins was identified by SDS-PAGE.

[0029] (2) Add cell lysis buffer (10 mmol / L PBS pH 7.4, 0.5 mol / L NaCl, 0.02 mol / L imidazole) and 2 mg / mL lysozyme, shake at 4 ℃ for 30 min, and then sonicate to fully lyse the cells; then add cell lysis buffer containing 6 mol / L urea, sonicate in an ice bath to help dissolve inclusion bodies, shake at 4 ℃ for 2 h; centrifuge at 12000 rpm at 4 ℃ for 30 min, and collect the supernatant.

[0030] (3) The protein with a purity of 96% was obtained by chelating and purifying it with nickel ions through the 6×HIS tag at the N-terminus of the recombinant protein.

[0031] Example 3: Preparation of HCV fusion antigen, including the following specific steps:

[0032] (1) Preparation of colloidal gold

[0033] Add ultrapure water and a clean rotor to a clean beaker. Using a magnetic stirrer, set the temperature to 250-350℃ and the stirring speed to boil the ultrapure water. After boiling, quickly add 10% chloroauric acid and stir until homogeneous. Then quickly add 10% trisodium citrate. Heat and stir for 5 minutes, then stir for 15 minutes with residual heat. Finally, move the mixture to room temperature and stir for 20 minutes until it cools naturally.

[0034] (2) Preparation of gold label

[0035] Take 20 mL of the above colloidal gold and place it in a beaker. While stirring, add 0.2 M K2CO3 to adjust the pH to 8.2 and continue stirring. Add a certain amount of HCV fusion antigen and continue stirring for 10 minutes. Add 0.2 mL of 10% BSA and continue stirring for 5 minutes. Centrifuge at 10,000 rpm for 30 minutes. Carefully aspirate the supernatant and collect the precipitate. Dilute the precipitate with colloidal gold dilution buffer (Tris 0.13 g, Casein 0.24 g, PVP 0.01 g, adjust the pH to 8.4, and bring the volume to 20 mL with ultrapure water. Dilute 1:1 and shake well. Measure the OD values ​​at 540, 560, and 600 nm (20 µL of immunogold added to 3 mL of ultrapure water).

[0036] (3) Gold spraying operation

[0037] Dilute with gold standard diluent to a total OD value of 75 (OD540 30*2.5µL / cm) or 125 (OD540 50*2.5µL / cm), and dry in an oven at 45℃ for 2 hours.

[0038] (4) Film drawing operation

[0039] The HCV-coated antigen was diluted to 0.3 mg / mL using a streak dilution buffer (Tris 0.05 g, NaCl 0.1 g, sucrose 0.1 g, pH adjusted to 8.6, and ultrapure water brought to a final volume of 20 mL), and then dried in a 45°C oven for 2 hours.

[0040] (5) Assembly

[0041] Assemble the above materials into a reagent strip while maintaining dehumidification.

[0042] (6) Detection

[0043] Add the sample to be tested to the sample pad, let it stand at room temperature for 15 minutes, and then interpret the results.

[0044] Performance testing

[0045] 1. Sensitivity Testing: HCV-RNA and magnetic microparticle chemiluminescence immunoassay were performed on 100 HCV antibody-positive samples. 42 samples were positive for HCV-RNA, and all 42 of these were also positive for magnetic microparticle chemiluminescence immunoassay. Based on the HCV-RNA result, the positive detection rate of the magnetic microparticle chemiluminescence immunoassay kit reached 100%. The limit of detection (NCU / mL) of HCV fusion antigen was determined using the Constellate hepatitis C virus antibody series serum (liquid) standard material (catalog number: GBW(E)090153). The results are shown in Table 1.

[0046] 2. Specific detection: 34 HAV (hepatitis A virus) positive serum samples, 148 HBV (hepatitis B virus) positive serum samples, 297 HIV (HIV) positive serum samples, and 21 TP (syphilis) positive serum samples were detected using a magnetic microparticle chemiluminescence detection kit. One HBV sample was positive. The detection results are shown in Table 1.

[0047] Table 1. Activity of purified protein

[0048]

[0049] As shown in Table 1, the negative concordance rate was 99.8%, proving that the recombinant HCV protein obtained by this invention has extremely high specificity and sensitivity.

[0050] 3. Thermal stability test of fusion protein

[0051] The HCV fusion antigen was stored at 37°C for 3, 5 and 7 days to test the stability of the test strips. The results are shown in Table 2.

[0052] Table 2 Results of HCV fusion antigen stability test

[0053]

[0054] As shown in Table 2, the differences between the samples after being placed at 37℃ for 3, 5 and 7 days were all within one G, indicating good stability.

[0055] 4. The recombinant protein of the present invention was compared with known HCV antigens in terms of sensitivity and specificity. The groups were set as follows: the fusion protein of the present invention (SEQ ID NO: 5), the Core protein expressed alone, the NS3 protein expressed alone, the NS5A protein expressed alone, and a physical mixture of the proteins (Core protein, NS3 protein and NS5A protein in a molar ratio of 1:1:1). The detection results are shown in Table 3.

[0056] Table 3. Results of HCV fusion antigen sensitivity and specificity detection

[0057]

[0058] As shown in Table 3, when detecting a series of borderline positive samples, the sensitivity of the experimental group (HCV fusion antigen) was significantly higher than that of the other control groups.

[0059] 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 recombinant fusion antigen protein of HCV, characterized in that, The recombinant fusion antigen protein comprises a Core antigen epitope of HCV, an NS3 antigen epitope region and an NS5A antigen epitope connected in series, wherein the amino acid sequence of the Core antigen epitope is shown as SEQ ID NO: 1, the amino acid sequence of the NS3 antigen epitope comprises SEQ ID NO: 2, and the amino acid sequence of the NS5A antigen epitope is shown as SEQ ID NO:

3.

2. The HCV recombinant fusion antigenic protein according to claim 1, characterized in that, The Core antigen epitope, the NS3 antigen epitope and the NS5A antigen epitope are connected by a peptide linker.

3. The HCV recombinant fusion antigenic protein according to claim 2, characterized in that, The peptide linker is a flexible linker, and the amino acid sequence of the peptide linker is shown as SEQ ID NO:

4.

4. The HCV recombinant fusion antigen protein according to claim 1, characterized in that, The amino acid sequence of the recombinant protein is shown as SEQ ID NO:

5.

5. The HCV recombinant fusion antigen protein according to claim 1, characterized in that, The N-terminus or C-terminus of the recombinant fusion antigen protein further comprises a purification tag, and the purification tag is a histidine tag.

6. A nucleic acid molecule encoding the HCV recombinant fusion antigen protein according to any one of claims 1-5.

7. The nucleic acid molecule of claim 6, wherein, The nucleic acid molecule comprises the nucleotide sequences shown as SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or a fusion sequence thereof connected by a linker sequence, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:

9.

8. A recombinant expression vector comprising the nucleic acid molecule according to claim 6, and the recombinant expression vector is pET28a (+).

9. A host cell, characterized in that, The host cell comprises the recombinant expression vector according to claim 9, and the host cell is Escherichia coli BL21 (DE3).

10. Use of the HCV recombinant fusion antigen protein according to any one of claims 1-5 in the preparation of an HCV antibody detection reagent.

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