A canine-derived monoclonal antibody for neutralizing canine parvovirus and its application

Abstract

The present invention relates to the technical field of canine viral disease treatment, and in particular to a canine-derived monoclonal antibody for neutralizing canine parvovirus and its application. The amino acid sequence of the heavy chain variable region of the canine-derived monoclonal antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2. The antibody provided by the present invention is completely derived from dogs. In addition to having better reactivity, the antibody will not cause immune rejection reaction of the body, so it can be repeatedly injected, and the neutralization titer is 0.97 μg / mL.

Description

Technical Field

[0001] The invention relates to the technical field of canine viral disease treatment, in particular to a canine-derived monoclonal antibody for neutralizing canine parvovirus and an application thereof. Background Art

[0002] Dogs are important companion animals for humans. Due to problems such as virus mutation, low immunization coverage, and interference from maternal antibodies, the clinical incidence of many viral infectious diseases, such as canine parvovirus disease, canine distemper, and canine coronavirus disease, remains high. Injection of monoclonal antibodies is currently one of the most important means of treating canine viral diseases in clinical practice. However, the monoclonal antibodies currently used in clinical practice are derived from mice. Although they have the effect of neutralizing viruses, as an exogenous protein, they often cause immune rejection reactions in dogs. In order to solve the above problems, some researchers have tried to genetically engineer anti-parvovirus monoclonal antibodies, such as preparing single-chain antibodies or genetically engineered chimeric antibodies. Although these antibodies have certain advantages, some of them are still derived from mice, and the side effects caused by heterology cannot be completely eliminated.

[0003] In recent years, single B cell antibody preparation technology has gradually been widely used, showing unique advantages and good application prospects. This technology mainly uses flow cytometry to sort out single antigen-specific B cells, and clones and expresses the light chain and heavy chain genes encoding antibodies in vitro. The monoclonal antibodies produced by this method ensure the natural pairing of light and heavy chain variable regions. Compared with traditional antibody preparation technology, it has the advantages of high efficiency, all-natural origin, and richer genetic diversity. At present, single B cell antibody preparation technology has become a popular method for preparing human antibodies and an important tool for modern life science research. However, this technology is currently mainly used in the research of human and mouse antibodies, and there has been no report on the preparation and application of this method for canine monoclonal antibodies.

[0004] The main treatments for canine parvovirus disease include specific treatments based on monoclonal antibodies and high-immune serum, as well as symptomatic auxiliary treatments such as antidiarrhea, antibacterial and anti-inflammatory, and fluid replacement. Since antibodies can directly bind to viruses specifically, prevent viral invasion and assist in killing virus-infected target cells through various pathways, they play a vital role in the clinical treatment of canine parvovirus disease. The monoclonal antibodies currently used in clinical practice are derived from mice. Although they have the effect of neutralizing viruses, since they are derived from heterologous animals, these antibodies, as exogenous proteins, often cause immune rejection reactions in dogs after entering the body; at the same time, due to the heterologous nature of the Fc fragment, these antibodies cannot effectively activate the activity of complement, macrophages, and killer cells in dogs, which greatly limits the role of mouse antibodies in dogs. These characteristics determine the shortcomings of heterologous antibodies, such as poor therapeutic effects and inappropriate repeated use of large doses. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a canine-derived monoclonal antibody for neutralizing canine parvovirus and its application.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a canine-derived monoclonal antibody for neutralizing canine parvovirus. The amino acid sequence of the heavy chain variable region of the canine-derived monoclonal antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2.

[0008] Preferably, the heavy chain CDR sequence includes a heavy chain CDR1 sequence, a heavy chain CDR2 sequence and a heavy chain CDR3 sequence;

[0009] The heavy chain CDR1 sequence is shown in SEQ ID No. 3;

[0010] The heavy chain CDR2 sequence is shown in SEQ ID No.4;

[0011] The heavy chain CDR3 sequence is shown as SEQ ID No.5.

[0012] Preferably, the light chain CDR sequence includes a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence;

[0013] The light chain CDR1 sequence is shown in SEQ ID No.6;

[0014] The light chain CDR2 sequence is GNN;

[0015] The light chain CDR3 sequence is shown in SEQ ID No.7.

[0016] The present invention also provides the use of the all-canine monoclonal antibody described in the above technical solution in the preparation of a drug or reagent for neutralizing canine parvovirus.

[0017] The present invention also provides the use of the all-canine monoclonal antibody described in the above technical solution in the preparation of a drug for treating canine parvovirus.

[0018] The present invention also provides a drug for treating canine parvovirus, comprising the all-canine monoclonal antibody described in the above technical solution and a pharmaceutically acceptable carrier.

[0019] The present invention also provides a kit for neutralizing canine parvovirus, comprising the all-canine monoclonal antibody described in the above technical solution.

[0020] The beneficial effects of the present invention are:

[0021] The present invention utilizes advanced single B cell antibody preparation technology, sorts single canine parvovirus (Canineparvovirus, CPV)-specific B cells, and amplifies, sequences, clones and expresses the antibody sequences encoded by them in vitro, thereby screening out all-canine anti-parvovirus monoclonal antibodies. This method is highly efficient and can ensure the natural pairing of the variable regions of the light and heavy chains of antibodies; on the one hand, since the antibody is completely derived from dogs, in addition to having better reactivity, the antibody will not cause the body's immune rejection reaction, so it can be repeatedly injected; more importantly, the Fc fragment of the antibody can also effectively activate the complement reaction and the activity of phagocytes and killer cells in the dog's body, helping the immune system to more effectively remove viruses and virus-infected target cells, so it will definitely have a better therapeutic effect. In addition, the antibody can be expressed and purified in large quantities by in vitro transfection of cells, so it has the conditions for large-scale production. In summary, the antibody is expected to replace the mouse parvovirus antibody used clinically, and has broad market application value and prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 Purified virus particles were observed for electron microscopy;

[0024] Figure 2 For the sorting of virus-specific B cells in PBMC;

[0025] Figure 3 The results of amplification of the variable regions of the heavy chain (A) and light chain (B) of canine antibodies (partial);

[0026] Figure 4 To construct the canine antibody heavy chain / light chain expression vector;

[0027] Figure 5 The expression of canine anti-CPV monoclonal antibodies (partial);

[0028] Figure 6 The reactivity of A6 antibody was verified by indirect immunofluorescence method;

[0029] Figure 7 The reactivity of antibody A6 was verified by ELISA;

[0030] Figure 8 This is the cytopathic effect diagram of the neutralization test, A. normal cells, B. diseased cells. DETAILED DESCRIPTION

[0031] IgG antibodies are "Y"-shaped molecules composed of two identical heavy chains (Heavy chain, H chain) and two identical light chains (Light chain, L chain). According to the degree of amino acid variation, both the heavy chain and the light chain can be divided into variable region (Variable region, V region) and constant region (Constant region, C region). The amino acid variation in the variable region is mainly concentrated in the hypervariable region. There are three hypervariable regions in the variable region of the heavy chain and light chain respectively. The hypervariable regions together form a three-dimensional spatial structure that complements and binds to the antigen epitope, so it is also called "complementarity determining region (CDR, complementarity-determining region). Since CDR is the region where the antibody directly binds to the antigen determinant cluster, it is generally highly specific and more important.

[0032] The present invention provides a canine-derived monoclonal antibody for neutralizing canine parvovirus. The amino acid sequence of the heavy chain variable region of the canine-derived monoclonal antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2.

[0033] SEQ ID No.1:

[0034] EVQLVESGGDLVKPGGSLRLSCETSGFTFYRYHMSWVRQAPGKGLQWV AYINSGGNSASYADAVKGRFTISSRDNAKNTLYLQMNSLRADDTAVYYCASGG AYYGSYYGPTDNFDYWGQGTLVTV.

[0035] In the present invention, the heavy chain CDR sequence includes a heavy chain CDR1 sequence, a heavy chain CDR2 sequence and a heavy chain CDR3 sequence; the heavy chain CDR1 sequence is shown in SEQ ID No.3; the heavy chain CDR2 sequence is shown in SEQ ID No.4; and the heavy chain CDR3 sequence is shown in SEQ ID No.5.

[0036] SEQ ID No.3: GFTFYRYH;

[0037] SEQ ID No.4: INSGGNSA;

[0038] SEQ ID No. 5: ASGGAYYGSYYGPTDNFDY.

[0039] SEQ ID No.2:

[0040] QSVLTQPASVSGSLGQKVTISCTGSSSNIGGYYVGWYQQLPGIGPRTVIY GNNYRPSGVPDRFSGSKSGSSATLTISGLQAEDEAEYFCSSWDDSLRDVVFGG GTHLTVL.

[0041] In the present invention, the CDR sequence of the light chain includes a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence; the light chain CDR1 sequence is shown in SEQ ID No.6; the light chain CDR2 sequence is GNN; and the light chain CDR3 sequence is shown in SEQ ID No.7.

[0042] SEQ ID No.6:SSNIGGYY;

[0043] SEQ ID No. 7: SSWDDSLRDVV.

[0044] The present invention also provides the use of the all-canine monoclonal antibody described in the above technical solution in the preparation of a drug or reagent for neutralizing canine parvovirus.

[0045] The present invention also provides the use of the all-canine monoclonal antibody described in the above technical solution in the preparation of a drug for treating canine parvovirus.

[0046] The present invention also provides a drug for treating canine parvovirus, comprising the canine monoclonal antibody and a pharmaceutically acceptable carrier as described in the above technical solution. The present invention does not specifically limit the type of the drug and its preparation method, and those skilled in the art can use conventional methods.

[0047] The present invention also provides a kit for neutralizing canine parvovirus, comprising the all-canine monoclonal antibody described in the above technical solution.

[0048] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] 1. Experimental dog immunization

[0051] Two healthy beagle dogs aged one month were purchased and immunized four times with canine parvovirus live vaccine (Intervet, USA), with an interval of 21 days between each immunization.

[0052] 2. Isolation of virus-specific B cells and amplification of antibody heavy and light chain variable region genes

[0053] (1) Preparation of CPV-Biotin: The CPV-LZ isolate (CPV-2c type, the NCBI accession number of its VP2 gene sequence is OQ869252) was isolated and preserved by our laboratory, and the virus was amplified in large quantities on F81 passage cells. The specific method is as follows: F81 cells were cultured and inoculated with the virus synchronously, and the cells were collected 60 hours after inoculation, and repeatedly frozen and thawed three times in a -80°C refrigerator. After centrifugation at 12000 rpm for 10 minutes, the supernatant was collected. After concentration with PEG6000, the virus was purified by sucrose density gradient ultracentrifugation and detected by electron microscopy ( Figure 1 ), the virus particles were labeled with a biotin labeling kit (Thermo Scientific, USA) and recorded as CPV-Biotin.

[0054] (2) Isolation of virus-specific B cells: After immunization, blood was collected from the jugular vein of the experimental dog, and peripheral blood mononuclear cells were separated using lymphocyte separation medium Ficoll Plus 1.077 (Solarbio). 7 Cells were resuspended in PBS solution at 100 cells / mL and stained: anti-dog IgM-FITC (Bio-Rad, USA), anti-canineCD21-PE (Bio-Rad, USA) and CPV-Biotin were added first, and the cells were incubated at 4°C for 30 minutes; after washing three times, anti-biotin-APC antibodies (Miltenyi Biotech) were added to the cell suspension as a secondary antibody and incubated at 4°C for 20 minutes; in order to better distinguish antigen-specific B cells, FMO controls were set up at the same time (except CPV-Biotin, cells were incubated with other antibodies in the same way). Subsequently, antigen-specific CD21 was sorted using a BD FACSAriaⅡ flow cytometer. + CPV + IgM-single B cell ( Figure 2 ), first circle the live cell population according to the FSC / SSC value ( Figure 2 A, P1 gate), then adherent cells were excluded based on the FSC-A / FSC-H plot, and single live cells were circled ( Figure 2 Then, based on the CD21 / IgM staining, we excluded naive B cells and circled CD21 + IgM-cells (mainly plasma cells and memory B cells, such as Figure 2 C, P3 gate), and finally according to FMO control ( Figure 2 D) and CPV labeling, circle the antigen-specific B cells ( Figure 2(E, gate P4). The cells in gate P4 were sorted into a 96-well plate pre-added with 10 μL single-cell lysis buffer (Invitrogen), with one cell per well; SuperScript TM IVVILO TM The cell cDNA was obtained by reverse transcription using MasterMix (Invitrogen) kit.

[0055] (3) Amplification of antibody heavy chain and light chain variable region genes: Based on the usage preference of canine antibody heavy chain variable region V genes (IGHV3 and IGHV4 were used most frequently, at 79.1% and 20.6%, respectively), upstream primers (HV3 and HV4) and downstream primers (common) were designed for IGHV3 and IGHV4, respectively; based on the usage preference of light chain variable region V genes (IGLVs:IGKVs were approximately 10:1; among IGLVs, IGLV1 was the most commonly used [77.2%], followed by IGLV8 [15.3%] and IGLV3 [5.9%]), kappa chain primers (not listed), IGLV1 and IGLV3 upstream primers (LV1 and LV3) and downstream primers (common) were designed, respectively. The primers are shown in Table 1. The nested PCR method was used to amplify the antibody heavy chain variable region sequence using the single cell cDNA in each well as a template, and the upstream and downstream primers of IGHV3 and IGHV4 were used to amplify the antibody light chain variable region sequence, respectively, using the κ chain primer (not listed), IGLV1 and IGLV3 upstream and downstream primers. The first round of PCR amplification system is as follows: 2×PhantaMaxMasterMix 25μl, 1μl of each of the outer upstream and downstream primers (OuterF / R), 5μl of single cell cDNA sample (template) and 18μl of ddH2O. The reaction procedure is as follows: pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 15s, annealing at 60℃ for 30s, extension at 72℃ for 50s, 30 cycles; extension at 72℃ for 10min. Using the first round PCR product as a template, the second round of amplification was performed using the inner upstream and downstream primers (InnerF / R), and the reaction procedure was the same as the first round. The electrophoresis diagram (partial) of the amplified product in each well is shown as follows. Figure 3 As shown, a total of 81 heavy chain PCR products were amplified from the 96 sorted single-cell cDNA samples, among which IGHV3:IGHV4=63:18; and 36 light chain products were amplified at the same time, among which IGLV1:IGLV3=35:1.

[0056] The following primers are named as SEQ ID No. 9 to 24.

[0057] Table 1 Primer sequences for amplifying antibody light chain and heavy chain variable regions

[0058] Forwardprimer(5'-3') Reverseprimer(5'-3') LV1-outer 8:GATGACCTYCAMCATGGSMTGGT 9:GCTTGGAGGGCTTGGTGGTCTC LV1-inner 10:GCCCAGTCTRTGCTGACTC 11:CGCTGGGGTAGAAGTCGCTG LV3-outer 12:TCAGCCTCCTGGCTCTCTG 13:GCTTGGAGGGCTTGGTGGTCTC LV3-inner 14:CACAGGTTTTGTGGCCTCCTATG 15:CGCTGGGGTAGAAGTCGCTG HV3-outer 16:TTGTCRCTATTTTAMAAGGTGTCC 17:GCCGGAATTCCAGGACACAG HV3-inner 18:GTGCAGCTGGTGGARTCTGG 19:AGCCTGACACCAGGCAGG HV4-outer 20:CAGCTCCCCTGGGTGTCC 21:GCCGGAATTCCAGGACACAG HV4-inner 22:CTGAACTCACACTGCAGGAGTC 23:AGCCTGACACCAGGCAGG

[0059] 3. In vitro expression and purification of antibodies

[0060] The paired light / heavy chain variable region PCR products were connected to the pUC19 vector (Sangon Biotech, China) and sent to Genewise Biotech Co., Ltd. for sequencing. The antibodies were named according to the position of the single cell in the 96-well plate, and the antibody light / heavy chain expression vectors were constructed. The specific method is as follows Figure 4 As shown: a signal peptide sequence was added to the 5' end of the antibody light / heavy chain variable region gene, and a λ light chain constant region sequence / IgG2 heavy chain constant region gene sequence and a His tag sequence were added to the 3' end. After codon optimization, the plasmid was cloned into the pcDNA3.4 vector and the company was commissioned to synthesize the plasmid. The recombinant heavy chain and light chain expression plasmids were added at a mass ratio of 2:3 using ExpiFectamine TM CHO transfection kit (Invitrogen, USA) was used to transfect into ExpiCHO-S TM After 5 days of suspension culture, the cell supernatant was collected and the antibody was purified using an AKTA protein purifier (GE Life Sciences). After eluting the antibody, it was dialyzed three times in a PBS solution and then concentrated with PEG6000 to obtain the purified antibody. The purified partial antibody was identified by non-denaturing / denaturing SDS-PAGE electrophoresis ( Figure 5 ), it can be seen that the antibody under non-denaturing conditions is about 170KD, and after denaturation, the antibody is divided into heavy chain (about 55KD) and light chain (about 30KD), indicating that the antibodies are correctly expressed and assembled. 22 antibodies were obtained by co-expression and purification, namely A1 / A4 / A5 / A6 / A8 / A9 / A10 / A11 / A12 / B2 / B6 / B10 / B11 / B12 / C12 / D1 / E1 / F1 / H8 / H10 / H11 / H12.

[0061] 4. Verification of antibody reactivity and neutralizing activity.

[0062] (1) Indirect immunofluorescence assay (IFA)

[0063] After F81 cells were subcultured, about 2×10 5Cells / well were plated in a 24-well plate and inoculated with CPV-LZ at 1 MOI. After 48 hours, the cells were fixed with paraformaldehyde for 15 minutes at room temperature; the fixative was discarded, 0.1% Tritonx-100 was added, and permeabilization was performed at room temperature for 5 minutes; the permeabilization solution was discarded, the cells were washed 3 times with PBS, and 5% BSA solution was added to block at room temperature for 2 hours; the blocking solution was discarded, the cells were washed 3 times with PBS, and purified A6 antibody (about 2ng / μL) was added to incubate at room temperature for 2 hours; the primary antibody was discarded, the cells were washed 3 times with PBS, and HRP-labeled anti-dog IgG antibody (Bio-Rad Laboratories) was added to incubate at room temperature for 1 hour. After washing the cells 4 times with PBS, the results were observed under a fluorescence microscope, and green fluorescence was observed in the cells, indicating that the A6 antibody had good reactivity with CPV-2 ( Figure 6 ).

[0064] The amino acid sequence of the heavy chain variable region of the A6 antibody is shown in SEQ ID No. 1, the CDR sequences of the heavy chain are the heavy chain CDR1 sequence, the heavy chain CDR2 sequence and the heavy chain CDR3 sequence, the heavy chain CDR1 sequence is shown in SEQ ID No. 3, the heavy chain CDR2 sequence is shown in SEQ ID No. 4, and the heavy chain CDR3 sequence is shown in SEQ ID No. 5;

[0065] The amino acid sequence of the light chain variable region of the A6 antibody is shown in SEQ ID No.2, the CDR sequences of the light chain are the light chain CDR1 sequence, the light chain CDR2 sequence and the light chain CDR3 sequence, the light chain CDR1 sequence is shown in SEQ ID No.6, the light chain CDR2 sequence is GNN, and the light chain CDR3 sequence is shown in SEQ ID No.7;.

[0066] (2) Enzyme-linked immunosorbent assay (ELISA)

[0067] The purified CPV-2 virus was diluted to 1 ng / μL with PBS and coated on a 96-well ELISA plate (100 μL / well) at 4°C overnight; the coating solution was discarded, the plate was washed 3 times with PBST, and 100 μL of 5% skim milk was added to each well for blocking at room temperature for 2 h; the blocking solution was discarded, the plate was washed 3 times with PBST, and the purified A6 antibody was first diluted to 10 μg / mL, and then diluted 2 times at a rate of 2 1 -2 10 The plate was then used as the primary antibody, 100 μL / well, incubated at room temperature for 2 h; the primary antibody was discarded, the plate was washed 3 times with PBST, and HRP-labeled anti-canine IgG antibody (Bio-Rad Laboratories) was used as the secondary antibody, incubated at room temperature for 1 h; the secondary antibody was discarded, the plate was washed 4 times with PBST, TMB substrate solution was added at 100 μL / well, color development was performed for 5 min; stop solution was added at 100 μL / well, and OD was measured with an enzyme reader 450 The absorbance value of each well at the wavelength. Two replicate wells were set for each antibody dilution, and a negative control (primary antibody plus PBS) was also set. The results are as follows Figure 7 As shown, the cut-off value = 2.1×negative control value, and values ​​greater than the cut-off value are judged as positive reactions, indicating that A6 antibodies can react with coated viral antigens above 0.02 μg / mL.

[0068] (3) Neutralization test

[0069] The neutralization ability of the antibody was determined at the cell level using a microneutralization test. The specific procedure was as follows: 100 μg / mL of purified A6 antibody was added to 2 1 -2 9 Perform a 2-fold dilution; take 50 μL of the diluted antibody and mix with an equal volume of 100 TCID 50 The virus solution was mixed and incubated at 37°C for 1 h. The virus-antibody mixture was added to an equal volume (100 μL) of freshly digested F81 cells (approximately 2×10 4 cells), were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator; an empty cell control and a 0.1 / 100 TCID 50 For virus-infected cell controls, 10 replicate wells were set for each antibody dilution and control. The cell lesions were observed every day during the culture process ( Figure 8 ), if the antibody has a neutralizing effect, the Reed-Muench method is used to calculate the amount of 100 TCID that can block 50% of the 50 The neutralizing titer of the antibody was obtained by dividing the initial and final concentrations of the antibody by the dilution of the antibody that caused the virus-induced cytopathic effect. The final calculated neutralizing titer of A6 was 0.97 μg / mL.

[0070] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A canine monoclonal antibody that neutralizes canine parvovirus, characterized in that: The amino acid sequence of the heavy chain variable region of the all-canine monoclonal antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The all-canine monoclonal antibody according to claim 1, characterized in that: The heavy chain CDR sequence includes a heavy chain CDR1 sequence, a heavy chain CDR2 sequence and a heavy chain CDR3 sequence; The heavy chain CDR1 sequence is shown in SEQ ID No. 3; The heavy chain CDR2 sequence is shown in SEQ ID No.4; The heavy chain CDR3 sequence is shown as SEQ ID No.

5.

3. The all-canine monoclonal antibody according to claim 1, characterized in that: The CDR sequence of the light chain includes a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence; The light chain CDR1 sequence is shown in SEQ ID No.6; The light chain CDR2 sequence is GNN; The light chain CDR3 sequence is shown as SEQ ID No.

7.

4. Use of the canine monoclonal antibody according to any one of claims 1 to 3 in the preparation of a drug or reagent for neutralizing canine parvovirus.

5. Use of the canine monoclonal antibody according to any one of claims 1 to 3 in the preparation of a drug for treating canine parvovirus.

6. A drug for treating canine parvovirus, characterized in that: The invention comprises the canine monoclonal antibody according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

7. A kit for neutralizing canine parvovirus, characterized in that: The invention comprises the fully canine monoclonal antibody as described in any one of claims 1 to 3.

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