A monoclonal antibody against tick-borne encephalitis virus and its application in the detection of tick-borne encephalitis virus.

Abstract

This invention discloses labeled antibodies and related biological materials, and their application in the detection of tick-borne encephalitis virus (TEV). The application includes any of the following: (1) preparing products for detecting TEV; (2) preparing products binding to the TEV E protein; (3) detecting TEV; and (4) binding to the TEV E protein. Compared with traditional HRP-labeled antibodies, this labeled antibody has higher detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to labeled antibodies and related biomaterials and their application in the detection of tick-borne encephalitis virus. Background Technology

[0002] Techniques such as ELISA and immunochromatography, which use viral antigens as detection targets, are important methods for detecting viral infections and play a crucial role in laboratory viral isolation and culture, vaccine titer determination, and efficacy evaluation. However, due to the low sensitivity of routine ELISA and immunochromatography, they are generally not used as the primary means of confirming infection. Tick-borne encephalitis virus (TBEV) belongs to the Flaviviridae family and Flavivirus genus. It is transmitted through tick bites and causes severe encephalitis. Establishing a sensitive and rapid viral detection method is of great significance for early treatment.

[0003] The detection of viral antigens mainly employs a double-antibody sandwich method. Capture antibodies capture and enrich viral antigens in the sample, and then detection antibodies are added to bind to the viral antigens. Signal collection and result interpretation are achieved by detecting the luminescent or colorimetric signals generated by the antibody conjugate. Chemical labeling methods, such as conjugating horseradish peroxidase (HRP), alkaline phosphatase, or biotin to the lysine residues of antibodies, are currently the most common antibody labeling methods. Detection can be achieved through enzymatic luminescence or chemiluminescence and is widely used in current immunological detection methods. However, for antibodies with multiple lysine residues in their binding region, labeling can easily affect the antibody's binding activity, especially in the double-antibody sandwich detection mode. The presence of multiple labeled enzyme molecules in the antibody binding region can create steric hindrance, preventing the detection antibody's Fab arms from fully binding to the viral antigen, thus reducing detection sensitivity.

[0004] Currently, there are no commercially available tick-borne encephalitis virus-specific monoclonal antibodies or labeled antibodies available for direct use, nor are there publicly available TBEV antibody gene sequences or preparation techniques for reference. Summary of the Invention

[0005] Currently, there are no commercially available tick-borne encephalitis virus (TBEV) specific monoclonal antibodies or labeled antibodies available for direct use, nor are there publicly available TBEV antibody gene sequences or preparation techniques for reference. This patent utilizes monoclonal antibody hybridoma technology combined with genetic engineering antibody technology to prepare a high-affinity detection antibody and obtain its gene sequence, which can be used for genetic engineering construction and in vitro expression.

[0006] This invention provides the use of an antibody or luciferase-labeled antibody in any of the following: the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the antibody or the luciferase-labeled antibody are as shown in positions 76-89, 107-113, and 146-154 of SEQ ID No. 6, respectively; and the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody or the luciferase-labeled antibody are as shown in positions 76-83, 101-108, and 146-158 of SEQ ID No. 8, respectively.

[0007] (1) Prepare products for detecting tick-borne encephalitis virus;

[0008] (2) Preparation of a product that binds to tick-borne encephalitis virus E protein;

[0009] (3) Detection of tick-borne encephalitis virus;

[0010] (4) Binds to the E protein of tick-borne encephalitis virus.

[0011] Alternatively, according to the above applications,

[0012] The amino acid sequence of the light chain variable region is shown in positions 32-164 of SEQ ID No. 6;

[0013] The amino acid sequence of the heavy chain variable region is shown in positions 32-169 of SEQ ID No. 8;

[0014] The amino acid sequence of the luciferase is shown in positions 506-675 of SEQ ID No. 8.

[0015] The light and / or heavy chains of the aforementioned luciferase-labeled antibodies may further include an IL-6 signal peptide, a flexible peptide, and / or luciferase. For example, the heavy chain of the aforementioned luciferase-labeled antibody may sequentially include an IL-6 signal peptide sequence, an antibody heavy chain, a flexible peptide, and luciferase.

[0016] Alternatively, according to the above applications,

[0017] The light chain amino acid sequence of the antibody or the luciferase-labeled antibody is shown in SEQ ID No. 6;

[0018] The heavy chain amino acid sequence of the antibody is shown in positions 32-499 of SEQ ID No. 8;

[0019] The heavy chain amino acid sequence of the luciferase-labeled antibody is shown in SEQ ID No. 8.

[0020] The present invention also provides a kit for detecting tick-borne encephalitis virus, comprising the above-mentioned antibody or luciferase-labeled antibody.

[0021] Optionally, the kit described above may also include a luciferase substrate and / or a negative control without tick-borne encephalitis virus.

[0022] The antibodies or luciferase-labeled antibodies mentioned above are also within the scope of protection of this invention.

[0023] The present invention also provides related biological materials for the above-mentioned antibodies or luciferase-labeled antibodies, wherein the related biological materials are any one of the following:

[0024] c1) Nucleic acid molecules encoding the aforementioned antibodies or luciferase-labeled antibodies;

[0025] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0026] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2), such as pcDNA3.1-ch2F5L and / or pcDNA3.1-ch2F5H-Nluc prepared in the following examples;

[0027] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0028] c5) Recombinant cells containing the nucleic acid molecule described in c1), or recombinant cells containing the expression cassette described in c2), or recombinant cells containing the recombinant vector described in c3).

[0029] Optionally, based on the aforementioned relevant biological materials, the nucleic acid molecule described in c1) is any of the following:

[0030] d1) The nucleotide sequence is the DNA molecule shown in positions 94-816 of SEQ ID No. 5 and / or positions 94-1497 of SEQ ID No. 7;

[0031] d2) has 90% or more identity with the nucleotide sequence defined by d1) and encodes a DNA molecule containing the aforementioned antibody or luciferase-labeled antibody;

[0032] d3) Hybridizes under strict conditions to the nucleotide sequence defined by d1) or d2) and encodes a DNA molecule containing the aforementioned antibody or luciferase-labeled antibody.

[0033] Optionally, based on the aforementioned relevant biological materials, the nucleic acid molecule described in c1) is any of the following:

[0034] e1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 5 and / or SEQ ID No. 7;

[0035] e2) has 90% or more identity with the nucleotide sequence defined by e1) and encodes the DNA molecule of the antibody or luciferase-labeled antibody described above;

[0036] e3) hybridizes under strict conditions with the nucleotide sequence defined by e1) or e2) and encodes a DNA molecule containing the aforementioned antibody or luciferase-labeled antibody.

[0037] The application of the aforementioned biological materials in any of the following situations also falls within the scope of protection of this invention:

[0038] (1) Prepare products for detecting tick-borne encephalitis virus;

[0039] (2) Preparation of a product that binds to tick-borne encephalitis virus E protein;

[0040] (3) Detection of tick-borne encephalitis virus;

[0041] (4) Binds to the E protein of tick-borne encephalitis virus.

[0042] In this article, identity refers to the similarity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage.

[0043] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0044] The stringent conditions can be hybridization and washing of the membrane at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0045] The preparation method of the above-mentioned antibody or the above-mentioned luciferase-labeled antibody may include: introducing a recombinant vector containing a gene encoding the above-mentioned antibody or the above-mentioned luciferase-labeled antibody into a host cell to obtain recombinant cells, culturing the recombinant cells to obtain a culture supernatant, wherein the culture supernatant contains the above-mentioned antibody or the above-mentioned luciferase-labeled antibody.

[0046] In the above preparation method, the host cell can be a eukaryotic cell, such as COS7 cells, CHO cells, HEK293 cells, yeast cells, or insect cells. In one embodiment of the present invention, the host cell is a COS7 cell.

[0047] This invention employs monoclonal antibody hybridoma technology combined with genetically engineered antibody technology to prepare a high-affinity detection antibody and obtain its gene sequence for genetic engineering construction and in vitro expression. A highly sensitive fluorescent molecule is used as a signal marker, and a more efficient intracellular labeling strategy is employed to prepare the labeled antibody, resulting in higher binding activity. This genetically engineered labeled antibody exhibits highly sensitive detection performance in tick-borne encephalitis virus detection.

[0048] Biomarking can be used to directionally recombine marker molecules at the end of the constant region of an antibody gene. Through gene transfection and cell culture, the marker antibody can be directly expressed, thus completely avoiding the influence of the marker on the antibody binding activity and improving detection sensitivity. Therefore, biomarking is a more efficient method for preparing labeled antibodies.

[0049] Taking advantage of the high signal intensity and wide linear range of Nanoluciferase, this invention uses Nanoluciferase as a marker to prepare a labeled antibody, Nano-ch2F5, for detecting tick-borne encephalitis virus, which has higher detection sensitivity than traditional HRP-labeled antibodies. Attached Figure Description

[0050] Figure 1 Example 1: ELISA was used to detect the binding activity of ch2F5 antibody and some other 7 antibodies to TBEV antigen.

[0051] Figure 2 Example 1: ELISA detection of ch2F5 antibody binding specificity.

[0052] Figure 3 Example 1: Affinity detection of ch2F5 antibody and recombinant E protein.

[0053] Figure 4 This is a schematic diagram of the construction of an antibody expression vector.

[0054] Figure 5 This is for the preparation and identification of Nluc-ch2F5 antibody in Example 2 and the detection of different antigen binding activities in Example 3.

[0055] Figure 6 This is for sensitivity and specificity detection in Example 3. Detailed Implementation

[0056] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0058] 1. Plasmids, cells, proteins, inactivated viruses, and clinical samples

[0059] pcDNA3.1(+) plasmid (Thermo Fisher Scientific), COS7 cells (laboratory-preserved, optimized eukaryotic expression of the extracellular region of tick-borne encephalitis virus envelope glycoprotein and its effect in serological detection, Huo Naifan, Kang Xiaoping, Hu Yi, Li Yuchang, Li Jing, Zhang Yu, Ran Xin, Jia Jia, Cao Xuefeng, Yang Yinhui, Biotechnology Communications, Vol.27 No.3 May, 2016, 38-41), MW06 and MW05 monoclonal antibodies (gifted by Shanghai Maiwei, described in Wang S, Peng Y, Wang R, Jiao S, Wang M, Huang W, Shan C, Jiang W, Li Z, Gu C, Chen B, Hu X, Yao Y, Min J, Zhang H, Chen Y, Gao G, Tang P, Li G, Wang A, Wang L, Zhang J, Chen S, Gui X, Yuan Z, Liu D. Characterization of neutralizing antibody with prophylactic and therapeutic efficacy against SARS-CoV-2 in rhesus monkeys. Nat Commun, 2020, 11(1):5752. doi:10.1038 / s41467-020-19568-1.), Serum No. 84 (laboratory preservation), inactivated tick-borne encephalitis virus (optimization of eukaryotic expression of the extracellular region of tick-borne encephalitis virus envelope glycoprotein and its effect in serological detection, Huo Naifan, Kang Xiaoping, Hu Yi, Li Yuchang, Li Jing, Zhang Yu, Ran Xin, Jia Jia, Cao Xuefeng, Yang Yinhui, Biotechnology Communications , Vol.27 No.3 May, 2016, 38-41), other viruses (respiratory syncytial virus (RSV), adenovirus type 3 (ADV), yellow fever virus YF17D (YF), rubella virus (RV), Japanese encephalitis virus (JEV), parainfluenza virus type 2 (Pinf-2), influenza virus H1N1 subtype, influenza virus H7N9 subtype, West Nile virus (WNV), Hantavirus (HTNV), described in Tang Y, Wang Y, Li Y, Zhao H, Zhang S, Zhang Y, Li J, Chen Y, Wu X, Qin C, Jiang T, Kang X. An integrated rapid nucleic acid detection assay based on recombinant polymerase amplification for SARS-CoV-2. Viral Sin.2022 Feb;37(1):138-141.doi:10.1016 / j.virs.2022.01.006.Epub 2022 Jan 13.PMID:35234627;PMCID:PMC8755414.) Retained by the laboratory.

[0060] 2 reagents

[0061] Phusion Ultra Fidelity PCR Kit (New England Biolabs, Catalog No. #M0530L)

[0062] PureLink TM Quick Gel Extraction Kit&PCR Purification Combo Kit (Invitrogen by Thermo Fisher Scientific, Cat. No. K220001)

[0063] Restriction endonucleases EcoRI (catalog number #R3101S), Notl (catalog number #R3189S), AflII (catalog number #R0520V) (New England Biolabs)

[0064] T4 DNA Ligase (Promega, catalog number M1801)

[0065] DH5 competent cells (Bolmai Biotechnology, catalog number BC102-01)

[0066] Spin Miniprep Kit (50) (QIAGEN, Item No. 27104)

[0067] Lipofectamine TM 3000 Transfection Reagent (Thermol Fisher Scientific, Cat. No. L3000-015)

[0068] Highly Sensitive Rapid Coomassie Brilliant Blue Staining Kit Protein Stains H C510041 (Sangon Biotech (Shanghai) Co., Ltd., Catalog No. C510041-0010)

[0069] Meilunbio Precast Page Gel (meilunbio, item number MA0296)

[0070] PageRulerPrestained Protein Ladder 26616 (Thermo, Cat. No. 26616)

[0071] PureProteome Protein A / G Mix Magnetic Beads(Millipore,CAT:LSKMAGAG10)

[0072] Nano-Glo Luciferase Assay, 10ml (Promega, N1110)

[0073] High-sensitivity chemiluminescence detection kit (Kangwei Century, CW0049M)

[0074] 3 Instruments

[0075] Electrophoresis apparatus (Hoefer EPS 2A 200) horizontal electrophoresis tank

[0076] Ultraviolet gel transilluminator (Gel Doc™ XR+)

[0077] Mini-PROTEAN Tetra System (BIO-RAD) Vertical Electrophoresis System

[0078] Fully automated chemiluminescence detector (Bioteck product)

[0079] GloMax Microplate Luminescence Analyzer (Promega)

[0080] Example 1: Screening of ch2F5 antibodies

[0081] I. Preparation of ch2F5 antibody

[0082] The recombinant envelope protein (E protein, eukaryotic expression optimization of the extracellular region of the tick-borne encephalitis virus envelope glycoprotein and its effect in serological detection) of tick-borne encephalitis virus (TBEV) was optimized and its effect in serological detection was evaluated. The immunogen was emulsified with an equal volume of Freund's complete adjuvant and administered subcutaneously at multiple sites on the back of 12-week-old female Balb / c healthy mice (purchased from Spiford Biotechnology Co., Ltd.). The injection dose was 0.4 mL of immunogen per mouse. Female Balb-c mice were immunized three times, with each immunization occurring two weeks apart. Starting with the second booster immunization, blood was collected from the orbital sinus of mice on the third day after each immunization to determine antibody titers. Mice with the highest serum titers were selected, and their spleens were dissected to prepare spleen cell suspensions. These suspensions were then cultured in DMEM complete medium at 37°C for 5 days. Mouse myeloma cells SP2 / 0 were fused with spleen cells to prepare hybridoma cells capable of secreting anti-TBEV E protein antibodies. After monoclonal culture of the hybridoma cells, the expression supernatant of each cell line was collected, and the binding activity of the antibody to the natural TBEV virus antigen in the culture supernatant of each monoclonal cell was determined by ELISA.

[0083] The ELISA testing process is as follows:

[0084] TBEV inactivated virus was diluted 5-fold with PBS and then coated onto 96-well cell culture plates at 100 μL / well, incubated overnight at 4°C. The plates were then blocked with PBST solution containing 3% BSA and incubated at 37°C for 1 h. The plates were washed three times. Hybridoma antibody supernatant was diluted 100-fold, 400-fold, 1600-fold, 6400-fold, and 25600-fold, and added to the wells at 100 μL / well, incubated at 37°C for 1.5 h. The plates were washed three times. HRP-labeled goat anti-mouse IgG (Kangwei Century Co., Ltd.) was diluted 1:2500 with antibody dilution buffer and added to the wells at 100 μL / well, incubated at 37°C for 0.5 h. The plates were washed five times, and TMB substrate was added for color development for 15 min. 2M sulfuric acid was added as a stop solution, and the OD450 was measured using a multifunctional chemiluminescence immunoassay analyzer. The OD450 values ​​of different antibodies were compared; antibodies with higher OD450 values ​​indicated higher binding activity to the viral antigen. From 34 hybridoma cells to be screened, the ch2F5 antibody was selected to have higher binding activity. The ELISA results of the ch2F5 antibody with some of the other 7 antibodies are as follows: Figure 1 As shown.

[0085] II. Detection of antibody binding specificity

[0086] The binding specificity of ch2F5 antibody to TBEV viral antigen was detected using an ELISA method. Japanese encephalitis virus (JEV), dengue virus (DV), H1N1 influenza A virus, respiratory syncytial virus (RSV), adenovirus (ADV), Vero cell lysate, and BSA protein were used as irrelevant antigens to determine the binding specificity of ch2F5 antibody.

[0087] The ELISA method is similar to the ELISA detection procedure in "I. Preparation of ch2F5 antibody". The only difference is that the TBEV inactivated virus is replaced with JEV, DV, H1N1 influenza A virus, RSV, ADV, Vero cell lysate, BSA protein and TBEV inactivated virus respectively, and then ch2F5 antibody diluted 1:100 with PBST solution is added.

[0088] The results are as follows Figure 2 As shown, the ch2F5 antibody can only bind to TBEV inactivated virus (TBE), indicating that the antibody has good binding specificity.

[0089] III. Affinity analysis of ch2F5 antibody and recombinant E protein

[0090] The assay was performed at room temperature using the Gator™ label-free assay system (Gator Bio, CA, USA). All test proteins were diluted with Q Buffer (PBS solution at pH 7.4 containing 0.02% Tween 20, 0.2% BSA, and 0.05% NaN3). A blank probe was used as a negative control. E protein was released to 385 nM and pre-coated onto the His probe. Serially diluted 2-fold antibody (from 112 nM to 1.75 nM) bound to the S1-RBD protein on the probe surface. After the assay, the probe was regenerated with Gly-HCl buffer (pH 1.5). Affinity was calculated using the 1:1 (Rmax Local fit) Binding model in the Gator assessment software, which determined the dissociation constant.

[0091] The results are as follows Figure 3 As shown, the affinity dissociation constant KD value of ch2F5 antibody and recombinant E protein is 9.78 × 10⁻⁶. -10 (M) indicates that the ch2F5 antibody can bind to the target protein with high affinity.

[0092] Based on the combined affinity analysis and ELISA results, the ch2F5 antibody can bind to TBEV virus antigen with high affinity and high specificity, making it an ideal antibody for TBEV virus detection.

[0093] IV. ch2F5 antibody gene sequence

[0094] RNA was extracted from the 2F5 hybridoma cell line, and its antibody gene was amplified. The gene sequence of the light chain variable region of this murine monoclonal antibody ch2F5 was determined as shown in SEQ ID No. 1, the amino acid sequence of the light chain variable region was shown in SEQ ID No. 2, the gene sequence of the heavy chain variable region was shown in SEQ ID No. 3, and the amino acid sequence of the heavy chain variable region was shown in SEQ ID No. 4. The heavy chain type is IgG1, and the light chain type is κ chain.

[0095] By linking the variable region gene and the constant region gene of the ch2F5 antibody, a full-length antibody gene can be obtained for use in the expression of genetically engineered antibodies.

[0096] Example 2: Preparation and Identification of Nluc-ch2F5 Antibody

[0097] 1. Construction of the recombinant expression plasmid for Nluc-ch2F5 antibody:

[0098] Using pcDNA3.1(+) as the expression vector, light chain expression vectors and heavy chain expression vectors of Nluc-ch2F5 were constructed respectively.

[0099] A fusion gene consisting of an artificially synthesized IL-6 signal peptide sequence and an antibody light chain sequence encoding ch2F5 was used as the antibody light chain expression gene IL-6-ch2F5L. AflII (CTTAAG) and EcoRI (GAATTC) restriction enzyme sites were added to the 5' and 3' ends of the IL-6-ch2F5L gene sequence, respectively. A tandem fusion gene consisting of an artificially synthesized IL-6 signal peptide sequence, an antibody heavy chain sequence encoding ch-2B5, an EcoRI (GAATTC) restriction enzyme site, a 12-base flexible peptide, and the Nanoluciferase gene (Nluc) was used as the antibody heavy chain expression gene IL-6-ch2F5H-Nluc. AflII (CTTAAG) and NotI (GCGGCCGC) restriction enzyme sites were added to its 5' and 3' ends, respectively. The IL-6-ch2F5L, IL-6-ch2F5H-Nluc, and pcDNA3.1(+) plasmids were double-digested with restriction endonucleases, ligated with T4 ligase, and transformed into DH5α competent cells to obtain recombinant plasmids pcDNA3.1-ch2F5L and pcDNA3.1-ch2F5H-Nluc. The plasmids were then identified by double-enzyme digestion and sequencing. A schematic diagram of plasmid construction is shown below. Figure 4 As shown in Figure A, pcDNA3.1-ch2F5L is represented by Figure B, and pcDNA3.1-ch2F5H-Nluc is represented by Figure B. The results of the double enzyme digestion identification are as follows: Figure 5As shown in A-2, lane 1 contains the IL-6-2F5L fragment (801 bp) obtained by double digestion of the recombinant plasmid pcDNA3.1-ch2F5L with restriction endonucleases AflII and EcoRI. Lane 2 contains the IL-6-ch2F5H fragment (2028 bp) obtained by double digestion of the plasmid pcDNA3.1-ch2F5H-Nluc with restriction endonucleases AflII and NotI. Lane M1 is the DL2000 DNA marker, and lane M2 is the DL15000 DNA marker. Double digestion and sequencing results indicate that pcDNA3.1-ch2F5L and pcDNA3.1-ch2F5H-Nluc are vectors obtained by inserting IL-6-ch2F5L and IL-6-ch2F5H-Nluc into pcDNA3.1(+), respectively.

[0100] pcDNA3.1-ch2F5L is a vector obtained by replacing a small fragment between the pcDNA3.1(+)AflII and EcoRI restriction sites with the antibody light chain expression gene IL-6-ch2F5L. The nucleotide sequence of the antibody light chain expression gene IL-6-ch2F5L is shown in SEQ ID No. 5, where positions 1-87 are the IL-6 signal peptide sequence, positions 88-93 are the restriction site, and positions 94-816 encode the ch2F5 antibody light chain sequence. Positions 814-816 are the stop codon. In the ch2F5 antibody light chain sequence, positions 94-492 encode the light chain variable region, positions 226-267 encode the LCDR1 gene, positions 319-339 encode the LCDR2 gene, and positions 436-462 encode the LCDR3 gene. The vector expresses the antibody light chain, whose amino acid sequence is shown in SEQ ID No. 6, wherein positions 32-164 are the light chain variable region, positions 76-89 are LCDR1, positions 107-113 are LCDR2, and positions 146-154 are LCDR3.

[0101] pcDNA3.1-ch2F5H-Nluc is a vector obtained by replacing the small fragment between the AflII and NotI restriction sites of pcDNA3.1(+)AflII with the antibody heavy chain expression gene IL-6-ch2F5H-Nluc. The nucleotide sequence of the antibody heavy chain expression gene IL-6-ch2F5H-Nluc is shown in SEQ ID No. 7, where positions 1-87 are the IL-6 signal peptide sequence, positions 88-93 are restriction sites, positions 94-1497 are the sequence encoding the ch2F5 antibody heavy chain, positions 1498-1503 are restriction sites, positions 1504-1515 are the gene encoding the flexible peptide, and positions 1516-2025 are the Nanoluciferase gene, with positions 2026-2028 being stop codons. In the ch2F5 antibody heavy chain sequence, positions 94-507 encode the heavy chain variable region, positions 226-249 are the HCDR1 gene, positions 301-324 are the HCDR2 gene, and positions 436-474 are the HCDR3 gene. This vector expresses the antibody heavy chain, whose amino acid sequence is shown in SEQ ID No. 8. Positions 1-29 are the IL-6 signal peptide, positions 30-31 are the restriction enzyme sites, positions 32-499 are the ch2F5 antibody heavy chain sequence, positions 500-501 are the restriction enzyme sites, positions 502-505 are the flexible peptide sequence, and positions 506-675 are the Nanoluciferase sequence. In the ch2F5 antibody heavy chain sequence, positions 32-169 are the heavy chain variable region, positions 76-83 are HCDR1, positions 101-108 are HCDR2, and positions 146-158 are HCDR3.

[0102] Construction of Nanoluc-pcDNA3.1 expression plasmid:

[0103] Using pcDNA3.1(+) as the expression plasmid, the Nanoluc-pcDNA3.1 expression plasmid was constructed.

[0104] EcoRI (GAATTC) and NotI (GCGGCCGC) restriction sites were added to the 5' and 3' ends of the artificially synthesized Nanolucase gene, respectively. The Nanoluc and pcDNA3.1(+) plasmids were double-digested using restriction endonucleases, ligated with T4 ligase, and transformed into DH5α competent cells to obtain the recombinant plasmid Nanoluc-pcDNA3.1. The plasmid was then identified by double restriction digestion and sequencing. Plasmid construction is as follows: Figure 4 As shown in C.

[0105] The results of double enzyme digestion identification are as follows: Figure 5As shown in Figure A-1, lane 1 contains the recombinant plasmid Nanoluc-pcDNA3.1, which was digested with restriction endonucleases EcoRI and NotI to obtain the Nanoluc fragment (550 bp). Lane M1 contains the DL2000 DNA marker, and lane M2 contains the DL15000 DNA marker. The results of double digestion and sequencing confirm that Nanoluc-pcDNA3.1 is a vector obtained by inserting Nanoluc into pcDNA3.1(+).

[0106] 2. Expression and purification of Nluc-ch2F5 antibody

[0107] COS7 cells were cultured in DMEM containing 10% FBS and prepared into 24-well plates when the cells reached 80%-90% confluence. Lipofectamine was then used to... TM The 3000 transfection reagent transiently transfected COS7 cells with recombinant plasmids pcDNA3.1-ch2F5L and pcDNA3.1-ch2F5H-Nluc, using two different transfection methods.

[0108] Option 1: 0.25 μg each of recombinant plasmids pcDNA3.1-ch2F5L and pcDNA3.1-ch2F5H-Nluc, P3000 TM 1 μl, using Opti-MEM TM Bring the culture medium to a final volume of 25 μl. Option 2: 0.5 μg each of recombinant plasmids pcDNA3.1-ch2F5L and pcDNA3.1-ch2F5H-Nluc2F52F5, P3000. TM 2μl, using Opti-MEM TM The culture medium was brought to a final volume of 25 μl. Next, the two plasmid premixes and 25 μl of liposome premix (1.5 μl Lipofectamine) were added. TM Mix 3000 reagent and 25 μl Opti-MEM™ medium; add 3.1-0.5 μg of recombinant plasmid Nanoluc-pcDNA and 1 μl of P3000, and supplement with Opti-MEM medium to a final volume of 25 μl, then mix with 25 μl of liposome premix (1.5 μl Lipofectamine). TM Mix 0.5 μg empty vector pcDNA3.1, 1 μl P3000 reagent, and 25 μl Opti-MEM medium. Add the 0.5 μg empty vector pcDNA3.1 and 1 μl P3000 reagent to a final volume of 25 μl using Opti-MEM medium. Then mix with 25 μl liposome premix (1.5 μl Lipofectamine). TMMix 3000 ml of reagent and 25 μl of Opti-MEM medium; incubate at room temperature for 15 min, then add to a 24-well plate and incubate at 37°C in a 5% CO2 incubator. The recombinant plasmid Nanoluc-pcDNA3.1 was used as a negative control, and the empty vector pcDNA3.1 was used as a blank control.

[0109] Cell expression products were collected at 24h, 48h, 72h, and 96h post-transfection. After centrifugation, 20 μL of supernatant was collected and 50 μL of Nanoluc substrate (Nano-Glo Luciferase Assay, Promega, catalog number N1110) was added. The fluorescence intensity was measured using a GloMax microplate luminescence detector. The results are as follows: Figure 5 As shown in Figure B, 2F5 (0.5 μg) represents the fluorescence intensity of the expression product from cells transfected using Protocol 1, 2F5 (1.0 μg) represents the fluorescence intensity of the expression product from cells transfected using Protocol 2, Nanoluc-pcDNA3.1 represents the fluorescence intensity of the expression product from the control Nanoluc-pcDNA3.1, and Blank represents the fluorescence intensity of the expression product from the control pcDNA3.1. The results indicate that the luciferase intensity reached 10 ppm 24 hours after plasmid transfection. 8 High levels of antibody expression have been observed.

[0110] 20 ml of the cell expression product transfected for 24 h was diluted 10-fold with PBS, and 20 mg of Protein A / G conjugated magnetic beads (PureProteome Protein A / G Mix Magnetic Beads, Millipore, CAT: LSKMAGAG10) (10 mg / ml) was added. The mixture was incubated at room temperature for 60 min at 80 rpm using a rotary mixer. After magnetic separation, the supernatant was collected, the magnetic beads were washed twice with PBST, and 2 ml of citrate buffer (pH 3.0) was added for elution. The mixture was vortexed for 5 min, magnetic separation was performed, and the supernatant was collected. The pH was adjusted to 7.0 by adding 1 M Tris buffer.

[0111] 3. Protein gel electrophoresis detection of recombinant antibodies

[0112] Prepare 10-well, 0.75 mm thick, 12.5% ​​separating gel protein gels. Add 5× protein loading buffer to the samples (i.e., the supernatant of untreated cell expression products and the supernatant of cell expression products purified with protein A / G magnetic beads), boil for 10 min, cool, and then load the samples. The loading volume of samples and standard protein markers (molecular weight 10–180 kDa) is 10 μl. During electrophoresis, set the stacking gel voltage to 80 V and the separating gel voltage to 120 V. After electrophoresis, wash the protein gel twice with 25% isopropanol and twice with double-distilled water for 15 min each time. Immerse the gel in Nanoluc substrate and expose it with a chemiluminescence imaging device for 10 min to acquire film information. Then wash the gel with 20 ml of double-distilled water with shaking for 5 min, repeat 3 times. Stain with a high-sensitivity rapid Coomassie brilliant blue staining kit for 30 min, wash with 20 ml of destaining solution with shaking for 15 min, repeat 3 times, and acquire information with a gel imaging device.

[0113] The results are as follows Figure 5 As shown in C-1, lane 1 represents the unpurified Nluc-ch2F5 band (i.e., the supernatant of the untreated cell expression product), and lane 2 represents the purified Nluc-ch2F5 band (i.e., the supernatant of the cell expression product purified using protein A / G magnetic beads). M represents the Protein Ladder, 10 to 180 kDa. The results show that the heavy chain and light chain protein bands are 75 kDa and 25 kDa, respectively, consistent with the expected target bands, indicating that the Nluc-ch2F5 antibody was successfully prepared in "2. Expression and Purification of Nluc-ch2F5 Antibody".

[0114] Luciferase staining treatment method:

[0115] Prepare the luciferase substrate (Nano-Glo In-Gel Detection System, Promega, catalog number N3020) to determine the volume of Nano-Glo In-Gel Detection Reagent required to completely cover the gel. In a separate container, dilute 10 parts Nano-Glo In-Gel buffer 10-fold with deionized water and mix by inversion. Then dilute Nano-Glo Luciferase Assay Substrate 500-fold with buffer and mix by inversion.

[0116] After protein electrophoresis, the protein gel was washed twice with 25% isopropanol and shaken for 15 minutes each time. After that, the gel was quickly washed three times with deionized water, and then placed in water and shaken for 15 minutes each time, twice. The deionized water was discarded, and the gel was immersed in a sufficient amount of luciferase substrate and incubated with shaking for 5 minutes. The gel was then exposed using a chemiluminescence imaging system for 10 minutes to collect the film information.

[0117] The results are as follows Figure 5 As shown in C-2, lane 1 is the unpurified Nluc-ch2F5 band (i.e., the supernatant of the untreated cell expression product), and lane 2 is the purified Nluc-ch2F5 band (i.e., the supernatant of the cell expression product purified using protein A / G magnetic beads). M is the Protein Ladder, 10 to 180 kDa. The results show that only one heavy chain protein band, with a size of 75 kDa, is present, which matches the expected target band, indicating that the Nluc-ch2F5 antibody was successfully prepared in "2. Expression and Purification of Nluc-ch2F5 Antibody".

[0118] 4. Preparation of HRP-ch2F5 antibody

[0119] The ch2F5 antibody (its light chain sequence is shown in SEQ ID No. 6, and its heavy chain sequence is shown in positions 32-499 of SEQ ID No. 8) uses EZ-Link. TM Maleimide-activated horseradish peroxidase kit (Thermo Scientific, catalog number 31494) was used to conjugate horseradish peroxidase. The SATA method was used to add thiol groups to the ch2F5 antibody. 20 μl of SATA solution was added to the ch2F5 antibody and incubated at room temperature for 30 min. Then, 200 μl of conjugation buffer was added and incubated at room temperature for 2 h. The antibody was desalted using a desalting column, and the flow-through was collected. The flow-through was incubated with activated HRP at room temperature for 1 h to obtain HRP-ch2F5 antibody. EDTA was removed from the antibody by dialysis, and then 50% glycerol was added before storage at -20°C.

[0120] Example 3: Detection of the binding of Nluc-ch2F5 recombinant antibody to tick-borne encephalitis virus

[0121] 1. Determination of the binding activity of Nluc-ch2F5 recombinant antibody to tick-borne encephalitis virus

[0122] Direct ELISA was used to express 2.0 μg / mL JEV protein (prepared in our laboratory, described in Zheng Yang, Kang Xiaoping, Li Yuchang, et al., Expression and purification of Japanese encephalitis virus EDⅢ protein and its application in array-ELISA method, Chinese Journal of Microbiology and Immunology, December 2013, Vol. 33, No. 12, 954-957) and tick-borne encephalitis virus E protein (described in the extracellular region of tick-borne encephalitis virus envelope glycoprotein, eukaryotic expression optimization and its effect evaluation in serological detection, Huo Naifan, Kang Xiaoping, Hu Yi, Li Yuchang, Li Jing, Zhang Yu, Ran Xin, Jia Jia, Cao Xuefeng, Yang Yinhui, Biotechnology Communications, Vol. 27 No. 3). May 2016, 38-41) and 3% BSA (Solepro, Beijing) were coated onto white 96-well plates at 100 μL / well and incubated overnight at 4°C. The plates were then blocked with PBST solution containing 3% BSA and incubated at 37°C for 1 h. Recombinant antigens S1-RBD, JEV, and 3% BSA were used as irrelevant antigens for specific analysis. Then, the Nluc-ch2F5 recombinant antibody (with a luciferase intensity of 10) prepared in Example 2 and purified using protein A / G magnetic beads was added. 6 RLU (100 μl), Nano luciferase as a negative control, 0.1% BSA-PBST as a blank control, incubated at 37°C for 1.5 h, then 50 μL of luciferase substrate (Nano-Glo Luciferase Assay, Promega, N1110) was added, and the values ​​were measured in a GloMax microplate luminescence detector.

[0123] The results are as follows Figure 5 As shown in D, specific binding of Nluc-ch2F5 to tick-borne encephalitis virus E protein can be detected, indicating that the obtained Nluc-ch2F5 has good binding activity to tick-borne encephalitis virus E protein (i.e., TBE in the figure).

[0124] 2. Sensitivity of Nluc-ch2F5 and HRP-ch2F5 antibodies for detecting tick-borne encephalitis virus.

[0125] A double-antibody sandwich ELISA was used. 2 μg / ml of ch2F5 (i.e., the ch2F5 antibody prepared in Example 1) was coated onto a white 96-well plate (100 μl / well), incubated overnight at 4°C, and washed three times. The plate was then blocked with PBST solution containing 3% BSA at 37°C for 1 h, and washed three times. Finally, 5-fold serially diluted inactivated tick-borne encephalitis virus (10...) was added. 5 PFU / ml-160 PFU / ml (5 concentrations in total) or serially diluted tick-borne encephalitis virus E protein (1 μg / ml-0.06 ng / ml, 4-fold serial dilutions, 8 concentrations in total), 10 4JEV inactivated virus (pfu / ml) and 3% BSA were used as negative controls, and PBST solution containing 0.1% BAS was used as a blank control. The mixture was incubated at 37°C for 1.5 h and washed 5 times. Nluc-ch2F5 (purified with protein A / G magnetic beads as prepared in Example 2) and HRP-ch2F5 (100 ng / ml) prepared in Example 2 were added, and the mixture was incubated at 37°C for 1.5 h and washed 5 times. 100 μL of luciferase substrate (Promega, N1110) and 100 μL of chemiluminescence substrate (high-sensitivity chemiluminescence detection kit, Kangwei Century, catalog number CW0049M) were added respectively, and the values ​​were measured in a GloMax microplate chemiluminescence detector. The cutoff value was calculated by adding 5 times the standard deviation to the mean of the negative control group. The S / C values ​​(S / C = experimental group luc value / cutoff value) of the two labeled antibodies against different concentrations of TBE and tick-borne encephalitis virus were calculated. An S / C > 1 was considered positive.

[0126] The results are as follows Figure 6 As shown in Figures A-1 and A-2, for the detection of tick-borne encephalitis virus E protein (TBE recombinant protein), both Nluc-ch2F5 and HRP-ch2F5 showed a minimum detectable concentration of 0.98 ng / ml for the TBE recombinant protein, indicating consistent sensitivity for the recombinant antigen. For the detection of inactivated TBEV virus, in the double-antibody sandwich mode, the minimum detectable concentration of Nluc-ch2F5 antibody was 800 pfu / ml, while that of HRP-ch2F5 antibody was 2000 pfu / ml (diluted 50-fold). The detection sensitivity of Nluc-ch2F5 antibody was significantly higher than that of HRP-ch2F5 antibody.

[0127] The antibody protein sequences of Nluc-ch2F5 and HRP-ch2F5 antibodies are completely identical, differing only in the labeling molecule and the labeling agent. The results of this example fully demonstrate that the C-terminal luciferase-labeled antibody Nluc-ch2F5, prepared using an intracellular biomarker method, can detect viral antigens more sensitively than the HRP-labeled ch2F5 antibody.

[0128] 3. Specificity of Nluc-ch2F5 antibody for detecting tick-borne encephalitis virus

[0129] Eight inactivated viruses (tick-borne encephalitis virus (TBEV), respiratory syncytial virus (RSV), adenovirus type 3 (ADV), yellow fever virus YF 17D (YF), rubella virus (RV), Japanese encephalitis virus (JEV), parainfluenza virus type 2 (Pinf-2), influenza virus H1N1 subtype, influenza virus H7N9 subtype, West Nile virus (WNV), and Hantavirus (HTNV)) were used as detection antigens. 4Using pfu / ml JEV inactivated virus and 3% BSA as negative controls, detection was performed using a double-antibody sandwich method. The specific steps are the same as in "2. Sensitivity of Nluc-ch2F5 antibody and HRP-ch2F5 antibody for the detection of tick-borne encephalitis virus".

[0130] The cutoff value was obtained by adding 5 times the standard deviation of the negative control group to the mean value. The test results were determined by calculating the S / C value (S / C = experimental group luc value / cutoff value) for each virus. The positive threshold was set at an S / C value > 1.

[0131] The results are as follows Figure 6 As shown in Figure B, except for tick-borne encephalitis virus, the S / C values ​​of other inactivated viruses are all below 1, indicating that the antibody has good specificity and no cross-reaction with other viruses.

[0132] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An antibody or luciferase-labeled antibody that binds to tick-borne encephalitis virus, wherein the amino acid sequence of the light chain variable region of the antibody or the luciferase-labeled antibody is as shown in SEQ ID No. 2, and the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody or the luciferase-labeled antibody are as shown in positions 76-83, 101-108 and 146-158 of SEQ ID No. 8, respectively.

2. The antibody or luciferase-labeled antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in positions 32-169 of SEQ ID No. 8; The amino acid sequence of the luciferase is shown in positions 506-675 of SEQ ID No.

8.

3. The antibody or luciferase-labeled antibody according to claim 1 or 2, characterized in that: The light chain amino acid sequence of the antibody or the luciferase-labeled antibody is shown in SEQ ID No. 6; The heavy chain amino acid sequence of the antibody is shown in positions 32-499 of SEQ ID No. 8; The heavy chain amino acid sequence of the luciferase-labeled antibody is shown in SEQ ID No.

8.

4. A kit for detecting tick-borne encephalitis virus, characterized in that: Includes the antibody or luciferase-labeled antibody as described in claim 1.

5. The reagent kit according to claim 4, characterized in that: It also includes a luciferase substrate and / or a negative control without tick-borne encephalitis virus.

6. The use of the antibody or luciferase-labeled antibody according to any one of claims 1-3 in the preparation of a kit for detecting tick-borne encephalitis virus.

7. The biomaterials related to the antibody or luciferase-labeled antibody as described in claim 1, characterized in that: The relevant biomaterial is any one of the following: c1) A nucleic acid molecule encoding the antibody or luciferase-labeled antibody as described in claim 1; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3); c5) Recombinant cells containing the nucleic acid molecule described in c1), or recombinant cells containing the expression cassette described in c2), or recombinant cells containing the recombinant vector described in c3).

8. The related biomaterial according to claim 7, characterized in that: c1) The nucleic acid molecule described is any one of the following: d1) The nucleotide sequence encoding the antibody light chain is the DNA molecule shown in positions 94-816 of SEQ ID No. 5, and the nucleotide sequence encoding the antibody heavy chain is the DNA molecule shown in positions 94-1497 of SEQ ID No. 7; The nucleotide sequence defined by d2) has more than 90% identity with that defined by d1) and encodes a DNA molecule of the antibody or luciferase-labeled antibody as described in claim 1.

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