Human-mouse chimeric antibody against dabie bandavirus np protein and application thereof

Abstract

A human-mouse chimeric antibody against Dabie bunyavirus Np protein and application thereof, a light chain variable region of the antibody has a nucleotide sequence of SEQ ID NO:1 and an amino acid sequence of SEQ ID NO:2, wherein a light chain hypermutation region sequence has SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; a heavy chain variable region has a nucleotide sequence of SEQ ID NO:6 and an amino acid sequence of SEQ ID NO:7, and a heavy chain hypermutation region has SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. The antibody can specifically bind to Dabie bunyavirus antigen (Np). The immunochromatography reagent based on CDs / SiO2 fluorescent nanospheres has the advantages of simplicity, rapidness, high sensitivity, strong specificity and the like, and is suitable for on-site rapid detection.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to a human-mouse chimeric antibody against Dabie Bandar virus Np protein and its application. Background Technology

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is an acute zoonotic disease caused by Dabie bandavirus (formerly known as severe fever with thrombocytopenia syndrome bunyavirus, DBV). Clinical manifestations of SFTS, particularly severe and fatal cases, include viral hemorrhagic fever (VHF) accompanied by systemic symptoms including thrombocytopenia and / or leukopenia, systemic inflammatory response syndrome (SIRS), gastrointestinal symptoms, liver dysfunction, disseminated intravascular coagulation, and multiple organ failure. The World Health Organization has listed SFTS as one of the emerging infectious diseases requiring priority research and intervention.

[0003] Currently, there are no vaccines or specific drugs approved for clinical use by the U.S. Food and Drug Administration or the China Food and Drug Administration. The main treatments for deep vein thrombosis (DBV) are symptomatic supportive care and treatment of complications. While ribavirin is recommended for clinical use in China, it has not been found to have significant efficacy in altering clinical outcomes. Cytokine storms and impaired immune responses, including innate, cellular, and humoral immunity, play important roles in the pathogenesis of DBV. To date, the pathogenesis of DBV infection is not fully understood, which significantly limits the clinical treatment of severe SFTS. Therefore, research on early diagnosis and therapeutic drugs for DBV infection has theoretical guiding significance for the treatment of severe SFTS.

[0004] Immunizing mice with anti-Dabiebanda virus antigen revealed that monoclonal antibodies provided immunoprotection upon viral challenge. This result suggests that monoclonal antibodies could serve as a convenient, rapid, and effective treatment for DBV infection, providing strong evidence for future development of monoclonal antibody therapies for SFTS. Therefore, developing human-mouse chimeric anti-Dabiebanda virus antibodies is crucial for their better application in clinical diagnosis and treatment. Summary of the Invention

[0005] Technical Problem Solved: This invention provides a human-mouse chimeric antibody against Dabie Bandar Virus antigen Np and its application. A monoclonal antibody with high affinity for DBV antigen Np was screened using monoclonal antibody technology. The amino acid and nucleotide sequences of the variable regions of the heavy and light chains, which impart unique properties to the antibody, were obtained. Furthermore, the constant regions of the heavy and light chains of the antibody were modified using a human-mouse chimeric method, ultimately resulting in a antibody with specific antigen-binding properties. Based on CDs / SiO2 fluorescent nanospheres, fluorescently labeled anti-DBV specific antibodies were prepared for the development of viral immunoassay reagents. These reagents offer advantages such as simplicity, speed, high sensitivity, and strong specificity, making them suitable for rapid on-site detection.

[0006] Technical solution: A human-mouse chimeric antibody against Dabie Bandar virus Np protein, wherein the light chain hypermutation region of the antibody has the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and the heavy chain hypermutation region has the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.

[0007] A human-mouse chimeric antibody against Dabiebanda virus Np protein, wherein the light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:2 and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:7.

[0008] The above vector is a secretory eukaryotic expression vector for the antibody IgG1 subtype.

[0009] The application of the above-mentioned antibodies in the preparation of antibody drugs for the diagnosis and treatment of DBV virus infection.

[0010] The application of the above-mentioned antibodies in the preparation of drugs for the diagnosis and treatment of fever with thrombocytopenia syndrome.

[0011] The antibody described above was modified with CDs / SiO2 fluorescent nanospheres.

[0012] Beneficial Effects: This invention provides a human-mouse chimeric monoclonal antibody hmNP105 with high protective, high specificity, and high affinity against Np. NP is a protein expressed by Dabie Bandar virus (DBV), therefore, the monoclonal antibody hmNP105 of this invention can be applied to research on the diagnosis, treatment, and prevention of plague. This invention uses the DBV antigen Np as the target molecule to prepare a murine monoclonal antibody 6B5, and based on NP105, prepares the human-mouse chimeric antibody hmNP105. Functional identification of the prepared murine monoclonal antibody NP105 and the human-mouse chimeric antibody hmNP105 was performed. Immunological detection showed that the murine monoclonal antibody and the human-mouse chimeric antibody can specifically bind to the Np antigen. Conjugating CDs / SiO2 fluorescent nanospheres to hmNP105 prepares a fluorescently labeled anti-DBV specific antibody for the development of viral immunoassay reagents, which has the advantages of simplicity, speed, high sensitivity, and strong specificity, making it suitable for rapid on-site detection. Attached Figure Description

[0013] Figure 1 The image shows the SDS-PAGE results of recombinant Np protein; M in the right figure represents the marker; 1 represents the purified Np protein. The protein purity is over 90%.

[0014] Figure 2 Amplification of the variable region gene for the NP-specific antibody against DBV; M is DNA Marker, 1 is the heavy chain variable region gene VH, and 2 is the light chain variable region gene VL.

[0015] Figure 3 Expression and purification of human-mouse chimeric anti-DBV antibody hmNP105; M is protein marker, 1 is the heavy chain H and light chain L of the antibody;

[0016] Figure 4 The ELISA results of the human-mouse chimeric anti-DBV antibody hmNP105 show that the hmNP105 antibody has a strong binding ability to the NP protein.

[0017] Figure 5 The results are from paper chromatography for DBV antigen detection. The sensitivity of this detection method can reach approximately 10-100 pg. Detailed Implementation

[0018] Example 1: Preparation of hm NP105 antibody

[0019] Using NP protein ( Figure 1 Mice were immunized to prepare the mouse monoclonal cell line NP105, and the NP105 hybridoma cell line was cultured to prepare the human-mouse chimeric antibody hm NP105.

[0020] 1) Amplification and validation of antibody variable region gene fragments:

[0021] NP105 antibody-treated hybridoma cells were cultured to the logarithmic growth phase, and total RNA was extracted from the cells using the Trizol-chloroform-isopropanol method. The dried total RNA was dissolved in 20 µL of water, and the OD260 / OD280 ratio was measured to be 1.9. 14 µL of RNA was used for reverse transcription, with the mRNA from the total RNA serving as a template, and OligodT... 15 Using primers, reverse transcription amplification was performed to obtain single-stranded cDNA.

[0022] Design 19 VH upstream primers, 17 Vκ upstream primers, 4 VH downstream primers, and 3 Vκ downstream primers:

[0023] Vκ5' upstream primer:

[0024] Vκ-1

[0025] 5'-GGG CCC AGG CGG CCG AGC TCG AYA TCC AGC TGA CTC AGC C-3'

[0026] Vκ-2

[0027] 5'-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCW CCC AGT C-3'

[0028] Vκ-3

[0029] 5'-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGM TMA CTC AGT C-3'

[0030] Vκ-4

[0031] 5'-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGY TRACAC AGT C-3'

[0032] Vκ-5

[0033] 5'-GGG CCC AGG CGG CCG AGC TCG AYA TTG TRA TGA CMC AGT C-3'

[0034] Vκ-6

[0035] 5'-GGG CCC AGG CGG CCG AGC TCG AYA TTM AGA TRA MCC AGT C-3'

[0036] Vκ-7

[0037] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTC AGA TGA YDC AGT C-3’

[0038] Vκ-8

[0039] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TYC AGA TGA CAC AGA C-3’

[0040] Vκ-9

[0041] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TTC TCA WCC AGT C-3’

[0042] Vκ-10

[0043] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG WGC TSA CCC AAT C-3’

[0044] Vκ-11

[0045] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTS TRA TGA CCC ART C-3’

[0046] Vκ-12

[0047] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTK TGA TGA CCC ARA C-3’

[0048] Vκ-13

[0049] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CBC AGK C-3’

[0050] Vκ-14

[0051] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TAA CYC AGG A-3’

[0052] Vκ-15

[0053] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CCC AGW T-3’

[0054] Vκ-16

[0055] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTG TGA TGA CAC AAC C-3’

[0056] Vκ-17

[0057] 5’-GGG CCC AGG CGG CCG AGC TCG AYA TTT TGC TGA CTC AGT C-3’

[0058] Vκ 3’ downstream primer

[0059] VκR1

[0060] 5’-AGA TGG TGC AGC CAC AGT TCG TTT KAT TTC CAG YTT GGT CCC-3’

[0061] VκR2

[0062] 5’-AGA TGG TGC AGC CAC AGT TCG TTT TAT TTC CAA CTT TGT CCC-3’

[0063] VκR3

[0064] 5’-AGA TGG TGC AGC CAC AGT TCG TTT CAG CTC CAG CTT GGT CCC-3’

[0065] VH 5’ upstream primer

[0066] VH 1

[0067] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR MAG CTT CAG GAG TC-3’

[0068] VH 2

[0069] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTB CAG CTB CAG CAG TC-3’

[0070] VH 3

[0071] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG CTG AAG SAS TC-3’

[0072] VH 4

[0073] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAR CTG CAA CAR TC-3’

[0074] VH 5

[0075] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAG CTB CAG CAR TC-3’

[0076] VH 6

[0077] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTY CAR CTG CAG CAG TC-3’

[0078] VH 7

[0079] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAC GTG AAG CAG TC-3’

[0080] VH 8

[0081] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAS STG GTG GAA TC-3’

[0082] VH 9

[0083] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AWG YTG GTG GAG TC-3’

[0084] VH 10

[0085] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAG SKG GTG GAG TC-3’

[0086] VH 11

[0087] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAM CTG GTG GAG TC-3’

[0088] VH 12

[0089] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG CTG ATG GAR TC-3’

[0090] VH 13

[0091] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG CAR CTT GTT GAG TC-3’

[0092] VH 14

[0093] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTR AAG CTT CTC GAG TC-3’

[0094] VH 15

[0095] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAR STT GAG GAG TC-3’

[0096] VH 16

[0097] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTT ACT CTR AAA GWG TST G-3’

[0098] VH 17

[0099] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTC CAA CTV CAG CAR CC-3’

[0100] VH 18

[0101] 5’-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAC TTG GAA GTG TC-3’

[0102] VH 19

[0103] 5'-GCT GCC CAA CCA GCC ATG GCC CTC GAG GTG AAG GTC ATC GAG TC-3'

[0104] VH 3' downstream primer

[0105] VH R1

[0106] 5'-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC CGT GGT-3'

[0107] VH R2

[0108] 5'-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC TGT GAG AGT GGT-3'

[0109] VH R3

[0110] 5'-CGA TGG GCC CTT GGT GGA GGC TGC AGA GAC AGT GAC CAG AGT-3'

[0111] VH R4

[0112] 5'-CGA TGG GCC CTT GGT GGA GGC TGA GGA GAC GGT GAC TGA GGT-3'

[0113] The Vκ5' upstream primer, Vκ3' downstream primer, VH 5' upstream primer, and VH 3' downstream primer were each dissolved to a final concentration of 100 pmol / µL, then mixed thoroughly in a 1:1 volume ratio, and the Vκ5' upstream primer, Vκ3' downstream primer, and Vκ3' downstream primer were amplified separately. H V L Gene amplification was performed under the following conditions: 95℃ for 4 minutes, 95℃ for 30 seconds, 60℃ for 40 seconds, and 72℃ for 45 seconds, for 30 cycles; a final extension at 72℃ for 10 minutes was performed, followed by electrophoresis recovery and purification of the amplified gene fragment (see...). Figure 2 The vector was ligated into the pMD-18T vector, transformed into E. coli DH5α, and sequenced to obtain the light chain and heavy chain variable region sequences, as follows:

[0114] The nucleotide sequence of VH is as follows:

[0115] GAGGTGAAGCTTCTCGAGTCTGGAGGGGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTTACTGGATGAGTTGGGTCCGGCAGGCTCCAGGGAAAGGACTAGAGTGGATTGGAGAAATTAATCCAGATAGTATGATAACCTATACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGACAACGCCAAAAATACGCTGTACCTGCAAATGAGCAAAGTGAGATCTGAGGACGCAGCCCTTTTTTACTGTGCAAGATGGAATGGGAATGACTACGGTCCTATTCTTGACTACTGGGGCCGAGGCACCACTCTCACAGTCTCCTCA

[0116] The nucleotide sequence of VL is:<�

[0117] GACATTGTGATGACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGTCGGTGGGTGCAAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATCGTATGCAAACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAA

[0118] 2) Design of PCR primers and gene amplification

[0119] Heavy chain amplification primers:

[0120] F: 5’- GGTGTCCACTCGCTAGATGTGCAGCTGCAGGAATCGGGACCT-3’

[0121] R: 5’- GCCCTTGGTGGATGCTGCAGAGACAGTGAC-3’ <00~0259>Light chain amplification primers:

[0123] F: 5'-ACAGACGCTCGCTGCCAAATTGTGCTCACTCAGTCTCCAG-3'

[0124] R: 5'-TGCAGCCACCGTACGTTTGATTTCCAGTTTGGTC-3'

[0125] 3) Amplify the heavy and light chains of hm NP105 antibody.

[0126] Using the variable regions of the heavy and light chains that were correctly linked to the pMD18-T vector as templates, the heavy and light chain genes of the human-mouse chimeric antibody were amplified using the upstream and downstream primers of the heavy and light chains mentioned above.

[0127] (1) PCR amplification of antibody heavy and light chain genes

[0128] (2) 2% agarose gel electrophoresis, observe the target band under ultraviolet light, and cut the gel for recovery.

[0129] (3) Purify the target DNA fragment using a gel recovery kit and elute with deionized water.

[0130] 4) Double digestion of IgG expression plasmid

[0131] The IgG expression plasmids pFUSE-CHIg-hG1 and pFUSE-CLIg-hk (purchased from Invivogen) contain the base coding sequences of the heavy and light chain (Kappa) constant regions of human IgG1.

[0132] (1) Double digestion of pFUSE-CHIg-hG1 and pFUSE-CLIg-hk template vectors

[0133] (2) 1% agarose gel electrophoresis, UV-cut gel recovery.

[0134] (3) Purify the target DNA fragment using a gel recovery kit and elute with deionized water.

[0135] 5) Infusion PCR recombinant expression plasmid

[0136] Transform competent bacteria with 5 µL of the reaction solution, plate them on plates with the appropriate antibiotics, and send clones for sequencing the next day. Preserve the bacterial strain of the correctly sequenced clones and expand the culture to extract plasmids.

[0137] 6) Expression of hm NP105 antibody

[0138] (1) Take 250µL of pFUSE-CHIg-hG1-hmNP105H (i.e., 50µg) into 1mL of Opti-MEM medium, take 250µL of pFUSE-CLIg-hk-hmNP105K (i.e., 50µg) into 1mL of Opti-MEM medium, and take 200µL of 293Fectin into 2.8mL of Opti-MEM medium. Let the above three mixtures stand at room temperature for 5min.

[0139] (2) After thoroughly mixing the two plasmid mixtures, add 500 µL of Opti-MEM medium and mix well. Then, directly add the 293Fectin transfection reagent mixture and mix well. Let stand for 20 min. During this period, treat the 293F cells by centrifuging them and resuspending them in 293F Expression Medium. Then, count the cells and calculate the cell viability ratio using Pan Blue. Take 90 × 10⁶ cells... 6 One cell was placed in a culture flask and brought to a final volume of 94 mL with 293F Expression Medium.

[0140] (3) After 20 minutes, add 6 mL of the DNA and 293Fectin complex to the prepared 293F cells.

[0141] (4) The cells were cultured in a shaker incubator under the following conditions: 8% CO2, 120 rpm, 37°C. The cell supernatant was collected after 6 days.

[0142] 7) Purification of hm NP105 antibody

[0143] The collected cell supernatant was filtered through a 0.22 μm filter membrane, along with the equilibration buffer and eluent. The cells were then purified using an AKATA purification system following standard Protein A purification procedures, with loading at 1 mL / min and elution at 1.5 mL / min.

[0144] The hm NP105 antibody was successfully expressed and purified. SDS-PAGE results are shown in the image. Figure 3 .

[0145] 8) hm NP105 purification antibody enzyme-linked immunosorbent assay

[0146] Dilute NP protein to 2 µg / mL with coating buffer (0.1 M carbonate buffer, pH 9.6) and coat ELISA 96-well plates. Add 100 µL to each well and incubate overnight at 4°C. Block with PBST (PBS containing 0.5% Tween 20) and 5% skim milk-wash buffer and incubate at 37°C for 2 h. After washing 5 times with PBST, add 100 µL of NP105 (2 µg / mL starting concentration, 15 serial dilutions) to each well and incubate overnight at 4°C. Add 100 µL / well of goat anti-mouse secondary antibody (Beijing Zhongshan Company) diluted 1:4000 to each well and incubate at 37°C for 1 h. Add 100 µL / well of peroxidase substrate chromogenic solution and stop the reaction with 2 M sulfuric acid after 15 minutes at room temperature. Detect colorimetrically using dual wavelengths of 450 nm / 690 nm.

[0147] See results Figure 4 The purified monoclonal antibody hm NP105 shows good binding activity to NP protein and can recognize DBV protein antigen.

[0148] Example 2: Labeling and Detection of hm NP105 Antibody

[0149] 1) Preparation and washing / purification of CDs / SiO2 fluorescent nanospheres

[0150] Hexadecylamine bromide and NaOH solution were added to water and stirred. Then, a mixture of TEOS and CDs was added under strong stirring and the reaction proceeded. The precipitate was collected by centrifugation. Citric acid monohydrate was dried in a high-temperature vacuum drying oven and then reacted with nitrogen-degassed N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane at high temperature to form a CDs solution. This solution was then centrifuged at high speed, and large particles at the bottom of the centrifuge tube were discarded. CDs / SiO2 fluorescent nanospheres were prepared by co-hydrolysis. Hexadecylamine bromide (CTAB, final concentration 0.02 mg / mL~20 mg / mL) and NaOH (final concentration 0.010 mg / mL~6 mg / mL) were added to 99 mL of water and stirred at 80°C for 10 min~2 h. Then, a mixture of TEOS (percentage of water volume, final concentration 0.01%~12%) and CDs (percentage of water volume, final concentration 0.001%~5%) was added under strong stirring and the reaction proceeded for 1~5 h. The precipitate was collected by centrifugation and washed several times with ethanol and H2O. The centrifuged precipitate was dispersed in a mixture of HCl and ethanol and stirred to extract the residual organic template. The extraction was repeated, and the product was washed until neutral. Finally, the obtained amino-terminated CDs / SiO2 fluorescent nanospheres were dispersed in H2O.

[0151] 2) Preparation of CDs / SiO2-DBV monoclonal antibody conjugate.

[0152] First, succinic acid was reacted with amino-terminated CDs / SiO2 fluorescent nanospheres to carboxylate them, yielding CDs / SiO2-COOH; then, the carboxyl groups on the surface of CDs / SiO2 were activated using EDC / NHS, DBV antibody was added, and the mixture was incubated at room temperature to obtain the CDs / SiO2-SFTSV antibody conjugate.

[0153] 3) Establishment of DBV fluorescence detection method

[0154] A rapid paper chromatography detection method was constructed using hmNP105 antibody as the capture antibody and CDs / SiO2-DBV antibody conjugate as the detection antibody. Through antigen-antibody binding, the antigen is successively captured by the T and C lines on the nitrocellulose membrane. The sensitivity of this detection method can reach approximately 10-100 pg. Figure 5 ).

Claims

1. A human-mouse chimeric antibody against Dabie Bandar virus Np protein, characterized in that, The light chain variable region of the antibody has the amino acid sequence shown in SEQ ID NO:2, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:

7.

2. The use of the antibody according to claim 1 in the preparation of an antibody drug for diagnosing DBV virus infection.

3. The use of the antibody according to claim 1 in the preparation of a drug for diagnosing fever with thrombocytopenia syndrome.

4. The antibody of claim 1 modified with CDs / SiO2 fluorescent nanospheres.

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