Canine coronavirus monoclonal antibodies and their applications and products

Abstract

This invention provides a canine coronavirus monoclonal antibody and its applications and products, relating to the field of biotechnology. The canine coronavirus monoclonal antibody provided by this invention has variable regions comprising: a complementarity-determining region (CDR1-VH) having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region (CDR2-VH) having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region (CDR3-VH) having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region (CDR1-VL) having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region (CDR2-VL) having the amino acid sequence shown in SEQ ID NO.8, and a complementarity-determining region (CDR3-VL) having the amino acid sequence shown in SEQ ID NO.5. This monoclonal antibody exhibits high specificity and affinity, and can be used for the development of high-quality diagnostic kits, possessing significant commercial value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a canine coronavirus monoclonal antibody and its applications and products. Background Technology

[0002] Since 2010, China's pet industry has entered a period of rapid development. With the increasing prominence of aging populations, changes in family structure, rising per capita income, and evolving pet ownership attitudes, the pet population has continued to expand, reaching countless households. Along with the development of the pet industry, in-vitro diagnostics for pets has gradually gained importance. This is not only related to the physical health of pets but also closely connected to the health of their owners. Therefore, the diagnosis of infectious diseases in pets has become particularly important.

[0003] Canine coronavirus (CCV) is a highly contagious virus that infects the digestive tract of dogs, causing gastroenteritis, which is particularly severe in puppies and young dogs. The virus is transmitted via the fecal-oral route. Normally, CCV causes only mild illness; however, when co-infected with CPV, the mortality rate of infected individuals increases significantly.

[0004] Currently available canine coronavirus detection kits have low sensitivity, requiring high concentrations of the virus to be detected, leading to missed detections and false negatives. To improve the sensitivity and specificity of canine coronavirus detection, the development of highly specific and highly affinity monoclonal antibodies is a prerequisite.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The first objective of this invention is to provide a canine coronavirus monoclonal antibody to solve at least one of the above-mentioned problems.

[0007] A second objective of this invention is to provide the application of the above-mentioned canine coronavirus monoclonal antibody in the preparation of canine coronavirus detection products.

[0008] A third objective of this invention is to provide an immunochromatographic test strip.

[0009] The fourth objective of this invention is to provide an ELISA detection kit.

[0010] The fifth objective of this invention is to provide a cell line.

[0011] In a first aspect, the present invention provides a canine coronavirus monoclonal antibody, wherein the variable region of the canine coronavirus monoclonal antibody comprises: a complementarity-determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.8, and a complementarity-determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.5.

[0012] As a further technical solution, the amino acid sequence of the heavy chain variable region VH of the canine coronavirus monoclonal antibody is shown in SEQ ID NO.6.

[0013] As a further technical solution, the amino acid sequence of the light chain variable region VL of the canine coronavirus monoclonal antibody is shown in SEQ ID NO.7.

[0014] Secondly, the present invention provides the application of the above-mentioned canine coronavirus monoclonal antibody in the preparation of canine coronavirus detection products.

[0015] As a further technical solution, the product includes immunochromatographic test strips and ELISA detection kits.

[0016] Thirdly, the present invention provides an immunochromatographic test strip, the immunochromatographic test strip comprising a base plate and a sample loading pad, a labeling pad, a detection pad and a sample absorption pad sequentially stacked on the base plate;

[0017] The labeling pad contains the canine coronavirus monoclonal antibody, which is labeled with a marker.

[0018] As a further technical solution, the marker includes at least one of colloidal gold, colored microspheres, or quantum dot microspheres.

[0019] Fourthly, the present invention provides an ELISA detection kit, using the canine coronavirus monoclonal antibody as the detection antibody.

[0020] As a further technical solution, the canine coronavirus monoclonal antibody is labeled with horseradish peroxidase.

[0021] Fifthly, the present invention provides a cell line expressing the aforementioned canine coronavirus monoclonal antibody.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The canine coronavirus monoclonal antibody provided by this invention has the advantages of high specificity and high affinity, and can be used for the development of high-quality detection kits, thus having significant commercial application value. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0025] Figure 1 Flowchart for the preparation of recombinant canine coronavirus N protein antigen;

[0026] Figure 2 The results of gel electrophoresis of canine coronavirus N protein antigen;

[0027] Figure 3 Flowchart for the preparation of monoclonal antibodies against canine coronavirus N protein;

[0028] Figure 4 Results of gel electrophoresis of canine coronavirus monoclonal antibody;

[0029] Figure 5 The results of gel electrophoresis of recombinant canine coronavirus monoclonal antibody;

[0030] Figure 6 The detection results are provided in Embodiment 4 of the present invention. Detailed Implementation

[0031] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0032] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be referred to as "VH," and the variable domain of the light chain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable region of the light or heavy chain consists of a framework region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The framework region of the antibody, that is, the framework region of the combination of the light and heavy chains, plays a role in locating and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0033] The “framework” or “FR” region refers to the region outside of those defined as CDRs of the antibody variable domain. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0034] Typically, the variable regions VL / VH of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0035] In this invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three highly variable regions of the heavy chain variable region, and correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three highly variable regions of the light chain variable region.

[0036] In a first aspect, the present invention provides a canine coronavirus monoclonal antibody, wherein the variable region of the canine coronavirus monoclonal antibody comprises: a complementarity-determining region CDR1-VH having an amino acid sequence as shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having an amino acid sequence as shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having an amino acid sequence as shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having an amino acid sequence as shown in SEQ ID NO.8, and a complementarity-determining region CDR3-VL having an amino acid sequence as shown in SEQ ID NO.5.

[0037] The sequence of the above variable regions is shown in Table 1:

[0038] Table 1

[0039] CDR1-VH GFNIKDTY SEQ ID NO.1 CDR2-VH IDLEDGYT SEQ ID NO.2 CDR3-VH VPYSSTYPFDY SEQ ID NO.3 CDR1-VL SSVSY SEQ ID NO.4 CDR2-VL STS SEQ ID NO.8 CDR3-VL QQRSSYPWT SEQ ID NO.5

[0040] The canine coronavirus monoclonal antibody provided by this invention has the advantages of high specificity and high affinity, and can be used for the development of high-quality detection kits, thus having significant commercial application value.

[0041] In some preferred embodiments, the amino acid sequence of the heavy chain variable region VH of the canine coronavirus monoclonal antibody is shown in SEQ ID NO. 6:

[0042] EVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDLEDGYTRYDPKLQGKATITVDTSSNTAYLQLSSLTSEDTAVYYCVPYSSTYPFDYWGQGTTLTVSS (SEQ ID NO. 6).

[0043] In some preferred embodiments, the amino acid sequence of the light chain variable region VL of the canine coronavirus monoclonal antibody is shown in SEQ ID NO.7:

[0044] QIVLTQSPAIMSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTVSRMEAEDAATYYCQQRSSYPWTFGGGTKLEIK (SEQ ID NO. 7).

[0045] In some preferred embodiments, the binding protein further includes a light chain constant region and a heavy chain constant region. The combination of the constant region and the variable region yields a complete antibody.

[0046] Secondly, the present invention provides the application of the above-mentioned canine coronavirus monoclonal antibody in the preparation of canine coronavirus detection products.

[0047] The canine coronavirus monoclonal antibody provided by this invention has good specificity and high affinity, and can be used to prepare products for canine coronavirus detection.

[0048] In some preferred embodiments, the products include, but are not limited to, immunochromatographic test strips and ELISA test kits, or other products known to those skilled in the art.

[0049] Thirdly, the present invention provides an immunochromatographic test strip, the immunochromatographic test strip comprising a base plate and a sample loading pad, a labeling pad, a detection pad and a sample absorption pad sequentially stacked on the base plate;

[0050] The labeling pad contains the canine coronavirus monoclonal antibody, which is labeled with a marker.

[0051] The immunochromatographic test strip provided by this invention is easy to operate, reacts quickly, is highly sensitive and specific, and can quickly screen for coronavirus in pet dogs.

[0052] In some preferred embodiments, the marker includes, but is not limited to, at least one of colloidal gold, colored microspheres, or quantum dot microspheres.

[0053] Fourthly, the present invention provides an ELISA detection kit, using the canine coronavirus monoclonal antibody as the detection antibody.

[0054] The ELISA detection kit provided by this invention has high sensitivity and accurate detection.

[0055] In some preferred embodiments, the canine coronavirus monoclonal antibody is labeled with horseradish peroxidase.

[0056] Fifthly, the present invention provides a cell line expressing the aforementioned canine coronavirus monoclonal antibody.

[0057] This cell line can be used for the preparation of monoclonal antibodies against canine coronavirus.

[0058] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0059] Example 1

[0060] Preparation of recombinant canine coronavirus N protein antigen: Canine coronavirus N protein antigen was prepared via prokaryotic expression. The gene encoding the N protein is relatively conserved; therefore, this invention uses the N protein as a target antigen for coronavirus detection. The experimental procedure is as follows: Figure 1 As shown, in this invention, the synthesized gene sequence AB105373.1 was inserted into the vector PET-28a using Nde I and BamHI as restriction enzyme sites. The plasmid was then transformed into *E. coli* BL21 for induced expression. After bacterial cell lysis, the canine coronavirus N protein antigen was obtained by Ni affinity chromatography purification. The results are as follows: Figure 2 As shown.

[0061] Preparation of monoclonal antibody against canine coronavirus N protein: Experimental procedure as follows Figure 3 As shown, mice were immunized with recombinant canine coronavirus N protein to prepare canine coronavirus antigen. After three immunizations, mouse spleens were fused with mouse myeloma cells SP20 and coated with canine coronavirus antigen. ELISA titers were measured to screen for highly reactive antibody-producing cell lines. Subcloning was performed using limiting dilution to obtain monoclonal cell lines. Cells were injected into the peritoneal cavity of mice to prepare ascites. The ascites was extracted and purified by ammonium octanoate-sulfate precipitation to obtain crude antibody samples. These samples were then purified by protein A affinity chromatography to obtain antibody samples (labeled antibody 4). The gel electrophoresis results of the antibody samples are shown below. Figure 4As shown. In addition, antibody 1, antibody 2 and antibody 3 were also obtained during the antibody screening process. Experimental studies have shown that antibody 4 has the highest titer.

[0062] mRNA was extracted from the monoclonal cell line expressing antibody 4, reverse transcribed into cDNA, and upstream and downstream primers were designed to amplify the variable region sequence of the antibody. The complementarity-determining region sequence of this monoclonal antibody is shown in Table 1, the heavy chain variable region sequence is shown in SEQ ID NO. 6, and the light chain variable region sequence is shown in SEQ ID NO. 7.

[0063] Combining the variable region sequence of the antibody heavy and light chains with the constant region sequence, a sequence was synthesized and ligated into the pCDNA3.4 vector. The canine coronavirus monoclonal antibody was secreted and expressed via transient transfection of CHO cells. The cell fermentation supernatant was collected and purified by protein A affinity chromatography to obtain the recombinant canine coronavirus monoclonal antibody (denoted as recombinant antibody 4). The gel electrophoresis results of this recombinant canine coronavirus monoclonal antibody are shown below. Figure 5 As shown.

[0064] Example 2

[0065] The sensitivity of antibodies 1-3, antibody 4, and recombinant antibody 4 was determined by ELISA. The experimental steps are as follows:

[0066] Antigen coating: Dilute canine coronavirus antigen to a concentration of 0.5 ug / mL with CB coating buffer, add 100 μL to each well of a 96-well microplate, incubate overnight at 4°C, and then remove the solution.

[0067] Blocking: Prepare 5% skim milk powder with PBST, add 200ul to each well, and incubate at 37°C for 2 hours to block. Wash once with PBST.

[0068] Primary antibody: The canine coronavirus monoclonal antibody (antibody 1-4 and recombinant antibody 4) was serially diluted to 50 ng / mL with PBST, and then serially diluted 11 times. PBST was used as a negative control. 100 μL was added to each well and the mixture was incubated at 37°C for 30 min. The mixture was then washed 5 times with PBST.

[0069] Goat anti-mouse IgG-HRP: Dilute with PBST at a ratio of 1:10000, add 100 μL to each well, incubate at 37°C for 30 min, and wash 5 times with PBST;

[0070] Developing solution: TMB single-component developing solution, add 100 μL per well, and incubate at 37°C for 10 min;

[0071] Termination: Add 50 μL of stop solution to each well to terminate the reaction. Remove the ELISA plate and measure the absorbance at 450 nm using an ELISA reader. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075] Note: * indicates a parallel experimental group.

[0076] By comparing the titer results of antibody 4 obtained through screening with those of the other three antibodies (antibodies 1, 2, and 3), antibody 4 showed significantly higher detection sensitivity, with a detection limit of 0.4 ng / mL.

[0077] The results of antibody 4 and recombinant antibody 4 were compared, and the results were similar. The correct antibody sequence was confirmed by recombinant antibody expression and titer assay.

[0078] Example 3

[0079] An ELISA detection kit, using antibody 4 as the detection antibody, and the detection method is as follows:

[0080] Primary antibody coating: Dilute the primary antibody (antibody 3) to a concentration of 1ug / mL with CB coating buffer, add 100ul to each well of a 96-well microplate, incubate overnight at 4°C, and then remove the solution.

[0081] Blocking: Prepare 5% skim milk powder with PBST, add 200ul to each well, and incubate at 37°C for 2 hours to block. Wash once with PBST.

[0082] Samples: The antigen was serially diluted with PBST to 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.125 ng / mL, and 1.563 ng / mL. PBST was used as a negative control. 100 μL was added to each well and the mixture was incubated at 37°C for 30 min. The samples were then washed 5 times with PBST.

[0083] Secondary antibody: Antibody 4 labeled with HRP, diluted with PBST to 1ug / mL, added to 100ul per well, incubated at 37℃ for 30min, and washed 5 times with PBST;

[0084] Developing solution: TMB single-component developing solution, add 100 μL per well, and incubate at 37°C for 10 min;

[0085] Termination: Add 50 μL of stop solution to each well to terminate the reaction. Remove the ELISA plate and measure the absorbance at 450 nm using an ELISA reader. The results are shown in Table 3.

[0086] Table 3

[0087]

[0088]

[0089] Example 4

[0090] Using the ELISA detection kit provided in Example 3, and comparing it with commercially available canine coronavirus detection kits (colloidal gold kit 1 and colloidal gold kit 2), the canine coronavirus vaccine (Vesta 8 canine coronavirus vaccine) was tested. The results are as follows:

[0091] Take 10 μL of canine coronavirus vaccine, dilute it 10-fold with the diluent provided with the kit, and add it dropwise to the sample well of the reagent strip. Incubate at room temperature for 15 minutes, and observe the color development of the reagent strip. The results are as follows: Figure 6 As shown, the test strips of colloidal gold reagent kit 1 and colloidal gold reagent kit 2 showed color on line C but not on line T, resulting in a negative result.

[0092] When the canine coronavirus antigen is diluted to 50 ng / mL, 100 μL is added to the sample well of the test strip in the colloidal gold kit 1. The test strip shows a positive result when the C line and T line are visible.

[0093] The canine coronavirus vaccine was serially diluted 2, 4, 8, 16, 32, and 64 times and tested using the ELISA detection kit provided in Example 3. The results are shown in Table 4. It can be seen that the vaccine can still be detected even when diluted 64 times.

[0094] Table 4

[0095]

[0096]

[0097] The results showed that the ELISA prepared with antibodies 4 and 3 could detect diluted canine coronavirus vaccine, while many canine coronavirus test kits on the market can only detect recombinant antigens and do not react to canine coronavirus inactivated vaccine samples.

[0098] Example 5

[0099] An immunochromatographic test strip includes a base plate and a sample loading pad, a labeling pad, a detection pad, and a sample absorption pad stacked sequentially on the base plate; wherein the labeling pad contains antibody 4, which is labeled with colloidal gold; the detection pad has a detection line and a control line, the detection line is coated with a primary antibody (canine coronavirus antibody, which recognizes a different site than antibody 4), and the control line is coated with goat anti-mouse IgG polyclonal antibody.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody to canine coronavirus, characterized in that, The variable region of the canine coronavirus monoclonal antibody comprises a complementarity determining region CDR1-VH of an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH of an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH of an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL of an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL of an amino acid sequence as shown in SEQ ID NO. 8, and a complementarity determining region CDR3-VL of an amino acid sequence as shown in SEQ ID NO.

5.

2. The canine coronavirus monoclonal antibody according to claim 1, characterized by The amino acid sequence of the heavy chain variable region VH of the canine coronavirus monoclonal antibody is as shown in SEQ ID NO.

6.

3. The canine coronavirus monoclonal antibody according to claim 1, characterized by The amino acid sequence of the light chain variable region VL of the canine coronavirus monoclonal antibody is as shown in SEQ ID NO.

7.

4. Use of the canine coronavirus monoclonal antibody according to any one of claims 1-3 in the preparation of a canine coronavirus detection product. The product is an immunochromatography test paper or an ELISA detection kit.

5. An immunochromatographic test strip, characterized by, The immunochromatography test paper comprises a base plate and, sequentially and laminated on the base plate, a sample loading pad, a label pad, a detection pad, and a sample suction pad. The label pad contains the canine coronavirus monoclonal antibody according to any one of claims 1-3, which is labeled with a label.

6. The immunochromatographic test strip according to claim 5, characterized in that, The label comprises at least one of colloidal gold, colored microspheres, or quantum dot microspheres.

7. An ELISA test kit characterized in that, The canine coronavirus monoclonal antibody according to any one of claims 1-3 is used as a detection antibody.

8. The ELISA test kit according to claim 7, characterized in that, The canine coronavirus monoclonal antibody is labeled with horseradish peroxidase.

9. A cell line, characterized in that, The canine coronavirus monoclonal antibody according to any one of claims 1-3 is expressed.

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