Peroxidase with high catalytic efficiency and thermal stability and application of peroxidase in influenza virus detection

Abstract

The invention discloses peroxidase with high catalytic efficiency and thermal stability and application of the peroxidase in influenza virus detection, the peroxidase is obtained through bioinformatics screening and genetic engineering modification, has an amino acid sequence SEQ ID NO: 3, and is named as KAG7611237.1. Compared with the traditional horseradish HRP (SEQ ID NO: 1), the HRP provided by the invention has higher catalytic efficiency (Kmlt; 0.2 mM) and thermal stability (82% of activity is retained after treatment at 50 DEG C for 30 minutes); when the enzyme is applied to an influenza A / B virus detection kit, the detection sensitivity can be improved by 2.5 times (the detection limit is 200 copies / mL), and the total detection time is shortened to 40 minutes. Clinical verification shows that the sensitivity of the novel HRP kit to influenza A reaches 98%, the sensitivity of the novel HRP kit to influenza B reaches 96%, and the specificity of the novel HRP kit to influenza B reaches 100%.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a peroxidase with high catalytic efficiency and thermal stability and application thereof in influenza virus detection. Background Art

[0002] Influenza viruses belong to the Orthomyxoviridae family and the Orthomyxovirus genus. Influenza viruses cause influenza, commonly known as the flu, an acute respiratory illness classified as a Category C infectious disease. Clinical manifestations include high fever, cough, headache, muscle aches and weakness. Influenza viruses are divided into four types: A, B, C, and D. Influenza A and B are the two most common influenza viruses in clinical practice. Influenza A viruses are highly variable and can infect a wide range of hosts, including humans and animals. Common subtypes include H1N1 and H3N2. Influenza B viruses are relatively stable, infecting only humans. They are primarily divided into two lineages: Victoria and Yamagata. Both can cause seasonal influenza, but influenza A is more likely to cause large-scale epidemics. However, specific populations, such as children and the elderly, are susceptible to influenza B viruses and are prone to complications. Therefore, rapid typing of influenza A and B viruses facilitates the development of treatment and medication plans and epidemic control.

[0003] Commonly used detection methods for influenza viruses include rapid antigen detection, reverse transcription polymerase chain reaction detection, virus isolation and culture detection, and serological detection. According to the relevant regulations of the national "Influenza Diagnostic Criteria and Treatment Principles", virus isolation and culture detection is the gold standard for influenza viruses, but this method has problems such as complex operation, long time consumption, low sensitivity, and instability, and is not suitable for clinical detection applications. The reverse transcription polymerase chain reaction detection limit of influenza virus is mostly around 500 copies / mL, and the detection time is long, which is also not suitable for clinical detection applications. Serological testing identifies antigens through hemagglutination tests (HA) and hemagglutination inhibition tests (HI). Because standard serum is required, the cost is high. In addition, serological testing is time-consuming and is not suitable for clinical detection applications. Antigen-antibody detection methods are faster, but have the disadvantage of low specificity.

[0004] The principle of rapid antigen testing is based on antigen-antibody reaction. Influenza virus antigen refers to specific proteins on the surface of the virus. When the virus is present in respiratory samples, these antigens will combine with antibodies in the detection equipment under the specific catalysis of enzymes to form antigen-antibody complexes. The results of antigen-antibody binding are usually displayed through a color reaction. The tester judges whether influenza virus antigens are present based on the color results, and thus infers the infection situation. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies in the above-mentioned prior art and provide a peroxidase with high catalytic efficiency and thermal stability and its application in influenza virus detection. The peroxidase can improve the detection speed and sensitivity of influenza A (H1N1) and influenza B (H1N1) test kits, and the technology can be widely used in the rapid detection and diagnosis of influenza viruses.

[0006] To achieve the above object, the technical solution adopted by the present invention is: In the first aspect of the present invention, a peroxidase with high catalytic efficiency and thermal stability is provided, characterized in that its amino acid sequence is shown in SEQ ID NO: 3, and the peroxidase is named KAG7611237.1.

[0007] In a second aspect, the present invention provides a gene encoding the peroxidase described above, the sequence of which is shown in SEQ ID NO: 4.

[0008] The third aspect of the present invention provides a nucleic acid construct, characterized in that it comprises the gene as described above.

[0009] The fourth aspect of the present invention provides an expression vector, characterized in that it comprises the nucleic acid construct as described above.

[0010] The fifth aspect of the present invention provides a host cell, characterized in that it contains the expression vector described above.

[0011] A sixth aspect of the present invention provides a method for preparing the peroxidase as described above, comprising the following steps: a) culturing the host cell as described above under conditions suitable for expression; b) recovering the peroxidase.

[0012] In a seventh aspect, the present invention provides use of the peroxidase described above in detecting influenza viruses, wherein the influenza viruses include influenza A virus and influenza B virus.

[0013] In an eighth aspect, the present invention provides use of the peroxidase described above in preparing an influenza virus detection reagent, wherein the influenza virus includes influenza A virus and influenza B virus.

[0014] A ninth aspect of the present invention provides an influenza virus detection kit, characterized in that it comprises the peroxidase described above.

[0015] Preferably, the influenza virus includes influenza A virus and influenza B virus.

[0016] Preferably, the detection limit of the kit is ≤200 copies / mL.

[0017] Preferably, the total detection time of the kit is ≤40 minutes.

[0018] The beneficial effects of the present invention are: 1. The novel HRP (KAG7611237.1) provided by the present invention has significantly improved affinity for H2O2, with a Km value of <0.2 mM. Compared with traditional horseradish HRP (Km≈0.28 mM), the catalytic efficiency is increased by more than 40%, and the color development time of enzyme-linked immunosorbent assay (ELISA) can be greatly shortened.

[0019] 2. The new HRP provided by the present invention has an activity retention rate of ≥82% after treatment at 50°C for 30 minutes, while traditional HRP only retains 40%, making the kit more stable during transportation and storage, and especially suitable for use in high-temperature areas.

[0020] 3. The new HRP provided by the present invention reduces the detection limit of influenza A and B viruses to 200 copies / mL (the traditional method is 500 copies / mL), and the sensitivity is increased by 2.5 times, which can detect low viral load infection cases earlier.

[0021] 4. The new HRP kit provided by the present invention shortens the total detection time to 40 minutes (the traditional HRP kit requires 60 minutes), of which the color development reaction only takes 8 minutes (the traditional HRP kit requires 15 minutes), making it more suitable for emergency and rapid screening scenarios.

[0022] 5. The new HRP kit provided by the present invention has been validated in 200 clinical samples, with a sensitivity of 98% for influenza A, a sensitivity of 96% for influenza B, and a specificity of 100%, which is significantly better than the existing traditional HRP kit (sensitivity of 90% for influenza A, sensitivity of 88% for influenza B, and specificity of 94%). BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Multiple sequence alignment; Figure 2 :Protein identification result diagram; Figure 3 :Results of 5 newly discovered HRPs and commercial HRPs for detecting influenza A virus; Figure 4 : DLKcat prediction results of 5 newly mined HRPs; Figure 5 :Catalytic rate test results of traditional HRP and new HRP at different substrate concentrations; Figure 6 : HRP enzyme thermal stability evaluation results; Figure 7 : The standard curve obtained by fitting. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0025] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Materials and reagents used in the following examples are commercially available unless otherwise specified. In the following examples, where specific conditions are not specified, the experiments were conducted under conventional conditions or those recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products. Example 1

[0027] This embodiment provides a novel method for the excavation and preparation of HRP (peroxidase), comprising the following steps: In this example, the source of enzymes and gene library construction were as follows: Horseradish HRP (derived from the root of Armoracia rusticana) was retrieved from the National Center for Biotechnology Information (NCBI) database. The amino acid sequence of Fasta sequence corresponds to SEQ ID NO: 1. Using this sequence as a template, BLAST screening was performed to obtain 300 novel HRPs with 60-80% homology to horseradish HRP. A phylogenetic tree was constructed using the maximum likelihood model to divide the novel HRPs into four different groups, resulting in 32 samples. In order to select the most representative HRP, samples with a distant genetic relationship with other samples in this group were selected for simulation, such as Figure 1 It is a multiple sequence alignment diagram. Finally, 5 representative samples were obtained (see Table 1), and AlphaFold3 was used for structural simulation.

[0028] Table 1

[0029] The Escherichia coli JM109 and Escherichia coli involved in this embodiment E. coliBL21(DE3) was purchased from takara - Bioray Biotechnology (Beijing) Co., Ltd., pET-22b(+) plasmid was purchased from Novagen (the above-mentioned Escherichia coli BL21(DE3) strain is commercially available and does not require deposit for patent procedures). Blunting Kination Ligation (BKL) Kit and PrimeSTAR® HS DNA Polymerase were purchased from Bioray Biotechnology (Beijing) Co., Ltd., and Bradford protein concentration assay kit (detergent-compatible) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0030] 1. Gene cloning and recombinant plasmid construction involved in this example Based on the HRP gene sequence in the NCBI database (GenBank accession number: KAG7611237.1), specific primers were designed and NdeⅠ / XhoⅠ restriction sites were introduced: Upstream primer (SEQ ID NO: 13): 5′-CATATGATGCAGCTGACCCCGACCTTT-3′; Downstream primer (SEQ ID NO: 14): 5′-CTCGAGAATGACAGCAACAGAAGTGGTCGCGCCAGTCAAGCGATT-3′.

[0031] PCR amplification was performed using PrimeSTAR® HS DNA Polymerase. The reaction conditions were: initial denaturation at 98°C for 2 min; 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min / kb; and a final extension at 72°C for 5 min. The PCR product was verified by 1% agarose gel electrophoresis, and the target fragment was blunt-ended using a BKL Kit. The treated HRP gene fragment was ligated with the pET-22b(+) vector that had been treated in the same manner (overnight at 16°C) to construct the recombinant plasmid pET-22b(+)-HRP.

[0032] 2. Construction of the recombinant strain involved in this example The ligation product was transformed into competent E. coli JM109 cells, plated onto LB solid medium containing ampicillin, and cultured at 37°C for 16 hours. Single colonies were selected for colony PCR verification, and positive clones were sequenced to confirm the correct reading frame. The verified recombinant plasmid was transformed into the expression host E. coli BL21(DE3) to obtain the engineered strain BL21(DE3) / pET-22b(+)-HRP.

[0033] 3. Induced expression of the recombinant protein involved in this example Inoculate a single colony into 5 mL of LB liquid medium (containing 100 μg / L Amp) and culture at 37°C with shaking at 200 rpm to an OD600 of approximately 0.6. Transfer a 1% inoculum to TB medium and culture at 37°C until an OD600 of 0.8. Add 0.5 mM IPTG and induce expression at 25°C for 16 h. Harvest the cells by centrifugation at 8,000 × g for 10 min at 4°C and wash twice with PBS buffer (pH 7.4).

[0034] 4. Protein purification and identification involved in this example The cells were resuspended in lysis buffer (20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, pH 8.0) and ultrasonically disrupted (300 W, 3 s on / 5 s off, total time 30 min). After centrifugation at 12,000 × g for 30 min, the supernatant was collected, filtered through a 0.22 μm filter membrane, and loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. Contaminants were removed using elution buffers containing 20 mM and 50 mM imidazole, and the target protein was finally eluted with a buffer containing 250 mM imidazole. SDS-PAGE (12% separation gel) analysis showed a specific band of approximately 44 kDa, which was consistent with the theoretical molecular weight of HRP. Figure 2 Protein specificity was verified by Western blotting using an anti-His tag antibody; protein concentration was determined by the Bradford method, and the aliquots were stored at -80°C until use. Example 2

[0035] This example provides an application of a newly discovered HRP in influenza A virus detection, verifying the effectiveness of the newly discovered HRP in improving the detection speed and sensitivity of influenza A virus (FluA) test kits. The process includes the following steps: 1. Coating microplate Anti-FluA monoclonal antibody (Cat. No. Ab-FluA-01) was used as the capture antibody, diluted to 2 μg / mL in pH 7.4 PBS. 100 μL was added to each well and incubated at 37°C for 2 hours. After discarding the solution, 200 μL of blocking buffer containing 1% BSA in PBS was added to each well. The cells were blocked at 37°C for 1 hour and then tapped dry for later use.

[0036] 2. Sample Treatment with HRP-Conjugated Antibodies Prepare serial dilutions of FluA antigen standard (concentration range: 50-1000 copies / mL). Add 40 μL of sample diluent (pH 7.4 PBS containing 0.05% Tween-20) and 10 μL of clinical nasopharyngeal swab sample (final dilution 5-fold) to the sample wells to be tested.

[0037] 3.HRP-conjugated detection antibody Newly mined HRP was conjugated to anti-FluA detection antibody (molar ratio 1:5) using the sodium periodate method, purified, and diluted to 0.3 μg / mL, and 50 μL was added to each well.

[0038] 4. Incubation and Washing Incubate at 37°C for 30 minutes to form a capture antibody-antigen-HRP detection antibody complex. Wash five times with 30-fold diluted wash buffer (PBS containing 0.1% Tween-20), letting stand for 30 seconds each time, and pat dry.

[0039] 5. Color development and termination Color development reagent A (TMB solution, pH 5.0 citrate buffer) and color development reagent B (0.05% Solution) 50 μL each, develop color at 37℃ in the dark for 8 minutes. Add 50 μL stop solution , measure the OD 450 value immediately.

[0040] Reference Figure 3 , which are the results of detecting influenza A virus using the five newly mined HRPs screened in Example 1 and commercial HRP (YS09272B, Shanghai Yaji Biotechnology Co., Ltd.). It can be seen that KAG7611237.1 has the highest sensitivity and is screened as the optimal peroxidase, referred to as the new HRP. Example 3

[0041] This example provides an application of a newly discovered HRP in influenza B virus detection, and verifies the thermal stability improvement effect of the newly discovered HRP in an influenza B virus (FluB) kit, including the following steps: 1. Enzyme Activity Test 1-1. Reference Figure 4 , which are the DLKcat prediction results of the five newly mined HRPs. The prediction methods include: (1) Molecular docking: The HRP crystal structure (PDB 3M3Q) was modified in Chimera to remove water molecules and fill in missing residues, and then hydrogenated with the AMBERff14SB force field; (2) Ligand treatment: H2O2 and TMB (3,3',5,5'-tetramethylbenzidine) were optimized with Avogadro; (3) Gasteiger charge distribution docking parameters: AutoDock Vina set the search box center coordinates to the active site Surrounding 20×20×20 ų, exhaustiveness=32; (4) DLKcat prediction: RMSD of the input docking conformation <2.0 Å complex, active pocket electrostatic potential (APBS calculation), solvent accessible surface area (DSSP algorithm).

[0042] 1-2. The novel HRP (KAG7611237.1) screened in Example 2 and a conventional HRP (i.e., the commercial HRP YS09272B, Shanghai Yaji Biotechnology Co., Ltd.) were treated at 50°C for 0, 15, and 30 minutes, respectively, and the residual activity was measured. The reaction system consisted of TMB substrate (pH 7.4 PBS) and 0.05% H₂O₂. The OD₄50 change rate was measured at 37°C. The results are shown in Table 2: Table 2. Enzyme activity test

[0043] 1-3. Reference Figure 5 , are the catalytic rate test results of traditional HRP and new HRP at different substrate concentrations; reaction system: 200 μL containing 50 mM PBS (pH7.4), 0.1% TMB, substrate H2O2, enzyme amount gradient 0.1-10 μg Kinetic parameters: Km=0.28±0.03 mM (H2O2) calculated by Lineweaver-Burk double reciprocal method, .

[0044] From the above results, it can be seen that the enzyme activity and catalytic rate of the new HRP are significantly higher than those of the traditional HRP.

[0045] 2. Kit stability test 2-1. HRP enzyme stability evaluation at room temperature: The kits containing traditional HRP and new HRP were placed at 37°C for accelerated aging for 7 days, and the initial performance was compared. The results are shown in Table 3: Table 3. Kit stability test

[0046] 2-2. HRP enzyme thermal stability evaluation: The new HRP and traditional HRP were placed in a 50℃ heat accelerated state for 20 min at a concentration of 1 mg / mL, and the residual activity was compared. Figure 6 From the above results, it can be seen that the room temperature stability and thermal stability of the new HRP are better than those of the traditional HRP. Example 4

[0047] This example provides a gene encoding the newly mined HRP described in Example 1: (1) The nucleic acid sequence encoding HRP from the root of horseradish Armoracia rusticana is SEQ ID NO. 2; (2) The code comes from Arabidopsis suecica The nucleic acid sequence of HRP (GenBank accession number: KAG7611237.1) is SEQ ID NO. 4; (3) The code comes from Armoracia rusticana The nucleic acid sequence of HRP (GenBank accession number: KAG7539136.1) is SEQ ID NO. 6; (4) The code comes from Cardamine amara subsp. Amara The nucleic acid sequence of HRP (GenBank accession number: KAL1224891.1) is SEQ ID NO. 8; (5) The code comes from Sinapis alba The nucleic acid sequence of HRP (GenBank accession number: KAF8118471.1) is SEQ ID NO. 10; (6) The code comes from Raphanus sativus The nucleic acid sequence of HRP (GenBank accession number: XP_056853177.1) is SEQ ID NO.12. Test Example 1

[0048] 1. Clinical sample testing Two hundred nasopharyngeal swab samples from clinically suspected influenza patients (confirmed by RT-PCR: 100 FluA positive, 50 FluB positive, and 50 negative) were tested in parallel using the new HRP kit and the traditional HRP kit (the only difference between the new HRP kit and the traditional HRP kit is that the new HRP is used instead of the traditional HRP). Specifically: This kit uses a double-antibody sandwich assay for the rapid identification of human influenza A virus antigens (FluA-Ag) and human influenza B virus antigens (FluB-Ag) in specimens. Two purified antibodies are coated onto a microplate to form a solid-phase antibody. This antibody is then conjugated to an HRP antibody conjugate prepared using the periodate method to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the complex is developed with the substrate TMB (3,3',5,5'-tetramethylbenzidine). TMB is a chromogenic substrate based on the catalytic activity of peroxidase (HRP). The TMB chromogenic reagent utilizes HRP to catalyze the oxidation of colorless TMB in the presence of hydrogen peroxide (H2O2) to produce a blue soluble product (maximum absorption wavelength 650 nm). A buffer maintains enzyme activity, while a stabilizer prevents substrate autooxidation. Addition of a strong acid stop solution terminates the reaction, and the blue product protonates to a yellow color (maximum absorption wavelength 450 nm). The absorbance can then be quantified using a microplate reader. Signal intensity is positively correlated with target concentration. The absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader, and a standard curve was obtained by fitting with the absorbance as the ordinate and the target concentration as the abscissa. Figure 7 As shown, the linear equation is y=0.0012x+0.2007(R 2 =0.9996).

[0049] The sensitivity test results of the HRP kit for detecting different concentrations of FluA are shown in Table 4 below: Table 4. Detection results of the new HRP kit and the traditional HRP kit for different concentrations of FluA

[0050] It can be seen that the detection limit of the new HRP (200 copies / mL) is significantly lower than that of the traditional HRP (500 copies / mL).

[0051] The comprehensive performance comparison results of the new HRP kit and the traditional HRP kit are shown in Table 5 below: Table 5. Performance comparison between the new HRP kit and the traditional HRP kit

[0052] Comparison results show that the new HRP kit provided by the present invention has higher sensitivity and specificity for detecting influenza A (H1N1) and influenza B (H1N1) than traditional HRP kits; the detection speed is shortened from 60 minutes to 40 minutes. Therefore, the technology can be widely used in the rapid detection and diagnosis of influenza viruses.

[0053] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A peroxidase with high catalytic efficiency and thermal stability, characterized in that Its amino acid sequence is shown in SEQ ID NO:

3.

2. A gene encoding the peroxidase according to claim 1, characterized in that Its sequence is shown in SEQ ID NO:

4.

3. A nucleic acid construct, characterized in that Comprising the gene according to claim 2.

4. An expression vector, characterized in that Comprising the nucleic acid construct according to claim 3.

5. A host cell, characterized in that Comprising the expression vector according to claim 4.

6. A method for preparing the peroxidase according to claim 1, characterized in that: The following steps are involved: a) culturing the host cell according to claim 5 under conditions suitable for expression; b) recovering the peroxidase.

7. Use of the peroxidase according to claim 1 in detecting influenza viruses, wherein the influenza viruses include influenza A virus and influenza B virus.

8. Use of the peroxidase according to claim 1 in preparing an influenza virus detection reagent, wherein the influenza virus includes influenza A virus and influenza B virus.

9. An influenza virus detection kit, characterized in that It comprises the peroxidase according to claim 1.

10. The influenza virus detection kit according to claim 9, characterized in that The influenza virus includes influenza A virus and influenza B virus.

Images