Preparation method and application of monoclonal antibody of mouse anti-infectious spleen and kidney necrosis virus main capsid protein

Abstract

The invention discloses a preparation method and application of a monoclonal antibody for resisting main capsid protein of an infectious spleen and kidney necrosis virus of a mouse, and relates to the technical field of monoclonal antibodies. The monoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region, the heavy chain variable region contains complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, and the amino acid sequences of the complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 are respectively shown as SEQ ID NO.3-5; the light chain comprises a light chain variable region, and the light chain variable region comprises complementary determining regions CDR-L1, CDR-L2 and CDR-L3. The monoclonal antibody has excellent affinity to the main capsid protein of the infectious spleen and kidney necrosis virus, the titer of the monoclonal antibody is 1: 32,000, and the monoclonal antibody can be used for detecting the infectious spleen and kidney necrosis virus or the main capsid protein of the infectious spleen and kidney necrosis virus.

Description

Technical Field

[0001] This application relates to the field of monoclonal antibody technology, and in particular to a method for preparing and applying a monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus. Background Technology

[0002] With the increasing density of aquaculture, various diseases are frequently occurring, seriously hindering the healthy and sustainable development of the aquaculture industry. Among them, viral diseases, due to their rapid spread, high harm, and difficulty in prevention and control, often lead to decreased feed intake, abnormal behavior, and increased mortality in farmed fish, causing huge economic losses. Iridoviruses, as one of the most widespread and pathogenic viral pathogens in farmed fish in both freshwater and marine water, especially the infectious spleen and kidney necrosis virus (ISKNV) of the genus Leptocytovirus, have had a serious impact on the healthy and sustainable development of the aquaculture industry.

[0003] Currently, while polyclonal antibodies and corresponding ELISA methods based on whole-virus antigens have been developed for the detection and research of ISKNV, limitations such as strong cross-reactivity and unstable sensitivity still exist, restricting their widespread application in practice. Monoclonal antibody technology, due to its advantages of high specificity, good uniformity, and sustainable production, has been widely used in the field of virus diagnosis and prevention. However, monoclonal antibodies targeting the major capsid protein (MCP) of ISKNV are still rarely reported, and there is a lack of monoclonal antibodies in related technologies that can accurately identify the ISKNV-MCP epitope and are suitable for low-cost, high-throughput detection, hindering the development of rapid detection kits for this ISKNV virus and in-depth research on its infection mechanism.

[0004] Therefore, it is still necessary to develop a method for preparing and applying a highly specific murine monoclonal antibody against the major capsid protein of ISKNV. Summary of the Invention

[0005] This application provides a method for preparing and applying a monoclonal antibody against the major capsid protein of infectious spleen and kidney necrosis virus (MCP). Specifically, the invention expresses and purifies recombinant MCP protein using a prokaryotic or eukaryotic system, immunizes BALB / c mice, and uses hybridoma technology to screen cell lines that can stably secrete anti-MCP monoclonal antibodies, thereby obtaining a monoclonal antibody with high affinity and strong specificity.

[0006] To achieve the above objectives, a first aspect of this application provides a monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus, wherein the monoclonal antibody comprises a heavy chain and a light chain.

[0007] The heavy chain includes a heavy chain variable region, and the heavy chain variable region contains complementarity determination regions CDR-H1, CDR-H2 and CDR-H3;

[0008] The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.3;

[0009] The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.4;

[0010] The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.5;

[0011] The light chain includes a light chain variable region, and the light chain variable region contains complementarity determination regions CDR-L1, CDR-L2 and CDR-L3;

[0012] The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.7;

[0013] The amino acid sequence of CDR-L2 is LVS;

[0014] The amino acid sequence of CDR-L3 is shown in SEQ ID NO.8.

[0015] In some embodiments, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

[0016] In some embodiments, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.6.

[0017] A second aspect of this application provides a bispecific antibody comprising the aforementioned monoclonal antibody against the major capsid protein of mouse anti-infective spleen and kidney necrosis virus.

[0018] The third aspect of this application provides biological material related to a monoclonal antibody against the major capsid protein of mouse anti-infective spleen and kidney necrosis virus of the first aspect described above, comprising any one of A1) to A5);

[0019] A1) The nucleic acid molecule encoding the monoclonal antibody against the main capsid protein of the above-mentioned mouse anti-infective spleen and kidney necrosis virus;

[0020] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0021] A3) A recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2);

[0022] A4) Recombinant microorganisms containing the nucleic acid molecule described in A1), the expression cassette described in A2), or the recombinant vector described in A3);

[0023] A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), or the recombinant vector described in A3), wherein the recombinant cells do not contain propagation material.

[0024] In some embodiments, the nucleic acid molecule comprises a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

[0025] The fourth aspect of this application provides a method for preparing a monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus as described in the first aspect, which is obtained by culturing recombinant microorganisms or recombinant cells as described in the third aspect, followed by separation and purification.

[0026] A fifth aspect of this application provides an antibody-drug conjugate comprising the monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus described above.

[0027] A sixth aspect of this application provides a pharmaceutical composition comprising at least one of B1) to B3):

[0028] B1) Monoclonal antibodies against the major capsid protein of mouse infectious spleen and kidney necrosis virus as described in the first aspect above;

[0029] B2) Biomaterials as described in the third aspect above;

[0030] B3) Antibody-drug conjugates as described in the fifth aspect above.

[0031] The seventh aspect of this application provides the use of a monoclonal antibody against the major capsid protein of infectious spleen and kidney necrosis virus (ISBN) as described in the first aspect in the preparation of a reagent for detecting ISBN or the major capsid protein of ISBN.

[0032] This invention provides a highly specific murine monoclonal antibody against the major capsid protein of ISKNV, its preparation method, and its applications. Specifically, this invention expresses and purifies recombinant MCP protein in a prokaryotic or eukaryotic system, immunizes BALB / c mice, and uses hybridoma technology to screen cell lines that stably secrete anti-MCP monoclonal antibodies, thereby obtaining a monoclonal antibody with high affinity and strong specificity. The monoclonal antibody has a titer of 1:320,000, filling the gap in current ISKNV-specific immunoassay reagents. It provides a core antibody source for establishing sensitive and rapid immunological detection methods (such as colloidal gold test strips and ELISA kits), and also provides a key tool for viral protein function research, analysis of infection-host interaction mechanisms, and subsequent vaccine development. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0034] Figure 1 The results are for the identification of monoclonal antibody subtypes.

[0035] Figure 2 The images show the results of immunoblotting identification of monoclonal antibodies, with the left image showing the monoclonal antibody and the right image showing the negative control.

[0036] Figure 3 This is the result of the titer identification of the monoclonal antibody. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0038] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0039] Example 1

[0040] This embodiment prepared a monoclonal cell line that secretes antibodies against the major capsid protein of mouse infectious spleen and kidney necrosis virus, specifically including the following contents.

[0041] (1) Preparation of immunogen

[0042] Based on the published major capsid protein of infectious spleen and kidney necrosis virus (AAL98730.1), Wuhan Arora Biotechnology Co., Ltd. was commissioned to recombinantly express and purify the major capsid protein of spleen and kidney necrosis virus using a prokaryotic expression system. The specific sequence is as follows:

[0043] MGTYAVLTSEEREVVAQSSRSMLIEQCQVAPRVPVTPADNSLVHLDLRFSHPVKALFFAVKNVTHRNVQSNYTAASPVYVNNKVNLPLMATNPLSEVSLIYENTPRLH QMGVDYFTSVDPYYFAPSMPEMDGVMTYCYTLDMGNINPMGSTNYGRLSNVTLSCKVSDNAKTTAAGGGGNGSGYTVAQKFELVVIAVNHNIMKIADGAAGFPIL (SEQ IDNO.1).

[0044] (2) Preparation of immunized mice

[0045] Six- to eight-week-old female Balb / C mice were used for immunization. Specifically, 50 μg of the recombinant protein prepared above was diluted with PBS and rapidly mixed with the water-soluble adjuvant QuickAntibody-Mouse 5W at a 1:1 volume ratio. The mixture was then injected intramuscularly into the hind leg, with a total injection of 100 μL per mouse. A booster immunization was administered three weeks post-immunization using the same procedure.

[0046] (3) Cell fusion

[0047] Three days prior to fusion, 20 μg of the recombinant protein was injected intraperitoneally into immunized mice for shock immunization. The mice were then euthanized by cervical dislocation, soaked in 75% alcohol for three minutes, and their spleens were aseptically isolated. The spleens were then ground and passed through a 70-mesh cell sieve to prepare a single-cell suspension.

[0048] Take approximately 2 × 10 8 After digestion of SP2 / 0 cells, a suspension was prepared and mixed with a single-cell suspension of spleen cells in the same centrifuge tube. After centrifugation at 1000 rpm for 10 min, the cells were washed once with incomplete DMEM medium to thoroughly remove the supernatant. Then, 700 μL of preheated PEG 1500 was added, and the mixture was incubated at 37°C for 3 min. Next, 500 μL of incomplete DMEM medium was added and the mixture was incubated at 37°C for 3 min. Then, 10 mL of incomplete DMEM medium was slowly added, and the mixture was incubated at 37°C for 10 min. After centrifugation at 1000 rpm for 10 min, the cells were resuspended in complete DMEM medium containing HAT (hypoxanthine, aminopterin, thymidine) and seeded into ten 96-well plates. One week after confluence, the HAT-containing medium was replaced with complete DMEM medium containing HT (hypoxanthine, thymidine) to obtain hybridoma cells for later use.

[0049] (4) Screening

[0050] Once the hybridoma cells reach a density of 30% or higher, the culture supernatant of the hybridoma cells is collected for further screening. The specific screening steps are as follows:

[0051] S1. Dilute the recombinant protein with ELISA coating buffer and coat it in a 96-well plate. Coat each well with 100 μL containing 0.1 μg of recombinant protein. Then coat overnight at 4°C and wash three times with PBST for 5 min each time.

[0052] S2. After blocking with 1% skim milk at 37°C for 1 h, wash three times with PBST for 5 min each time.

[0053] S3. Using the supernatant of hybridoma cell culture as the primary antibody, incubate at 37°C for 1 h, then wash three times with PBST for 5 min each time.

[0054] S4. Using HRP-conjugated Goat-anti mouse IgG (H+L) antibody as the secondary antibody, incubate at 37°C for 1 h and then wash three times with PBST for 5 min each time.

[0055] S5. Add 100 μL of single-component TMB substrate solution to each well and react for 10 min.

[0056] S6. After adding 100 μL of stop solution to each well, measure the OD. 450 .

[0057] A positive result was defined as an absorbance value greater than 2.1 between the experimental well and the negative control well. After screening, a hybridoma cell line numbered 4D8 showed a strong positive result and met the screening criteria.

[0058] (5) Obtaining monoclonal cells

[0059] Approximately 50 hybridoma cells numbered 4D8 were seeded into a 96-well plate, and the wells containing single cells were labeled. When the cells grew to 1 / 3 of the bottom area of ​​the plate, they were screened using the method described above. Two rounds of subcloning were performed to obtain single-clone cells.

[0060] Example 2

[0061] This embodiment describes the identification and sequencing of monoclonal antibody subclasses of the monoclonal antibodies secreted by the screened monoclonal cells, specifically including the following content.

[0062] (1) Identification of monoclonal antibody subclasses

[0063] The ELISA experiment was performed according to the method described in Example 1, wherein the HRP-conjugated Goat-anti-Mouse IgG (H+L) antibody used as the secondary antibody was replaced with HRP-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgM, and IgA, respectively. The ELISA results were obtained by measuring OD... 450 Determine antibody subclasses.

[0064] The results of the identification are as follows Figure 1 As shown, the monoclonal antibody described in this invention is an IgG1 subclass.

[0065] (2) Determination of the variable region sequence of monoclonal antibodies

[0066] After expanding the above monoclonal cell culture, 1×10⁶ cells were taken. 6 Cells were lysed with 1 mL of Trizol and then sent to Wuhan Aurora Biotechnology Co., Ltd. for next-generation sequencing of the variable regions of the adversarial heavy chain and light chain. The IMGT method was used to predict the sequences.

[0067] Its heavy chain variable region sequence is as follows:

[0068] VKLQESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRRTPEKSLEWVATFSSVGTYTYYPDSVKGRFTISRDNAKNTLCLQMNSLRSEDTAMYFCVRNFDYWGQGTTVTVSS (SEQ ID NO. 2).

[0069] The CDR region sequence is as follows:

[0070] CDR-H1: GTFFSTYA (SEQ ID NO. 3).

[0071] CDR-H2: FSSVGTYT (SEQ ID NO. 4).

[0072] CDR-H3: VRNFDY (SEQ ID NO. 5).

[0073] Its light chain variable region sequence is as follows:

[0074] DIQLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWRSN (SEQ ID NO. 6).

[0075] The CDR region sequence is as follows:

[0076] CDR-L1: KSVSTSGYSY (SEQ ID NO. 7).

[0077] CDR-L2: LVS.

[0078] CDR-L3: QHIRELT (SEQ ID NO. 8).

[0079] Example 3

[0080] This embodiment describes the identification of monoclonal antibodies secreted by the above-screened hybridoma cell lines using Western blotting, which includes the following steps:

[0081] S1, take 1×10 6 The virus-positive protein sample was prepared by lysing the infectious spleen and kidney necrosis virus particles with loading buffer and heating in boiling water for 10 min. At the same time, the same number of mandarin fish ranavirus (MRV) was treated in the same way to prepare the control protein sample.

[0082] S2. Add the prepared protein sample to a 4-20% pre-prepared protein gradient gel and electrophores at a constant voltage of 160V for 45 min.

[0083] S3. After electrophoresis, use ice-free rapid transfer buffer to transfer the protein to a PVDF membrane at a constant current of 0.4A for 30 min.

[0084] After sealing with S4 and 5% skim milk for 1 hour, wash three times with TBST for 5 minutes each time.

[0085] S5. Use the culture supernatant of the hybridoma cell lines selected above as the primary antibody and the culture supernatant of SP2 / 0 cells as the control. After incubating overnight at 4°C, wash three times with TBST for 5 min each time.

[0086] S6. Goat-anti Mouse IgG (H+L) conjugated with HRP was used as a secondary antibody and incubated at room temperature for 45 min, followed by washing three times with TBST for 5 min each time.

[0087] S7. Perform color development according to the instructions for the High-sig ECL Western Blotting Substrate kit (Tanon, 180-5001).

[0088] The results are as follows Figure 2As shown, the left side represents the hybridoma cell line group, and the right side represents the SP2 / 0 cell group. The results show that the monoclonal antibodies secreted by the hybridoma cell lines screened in this invention only recognize the major capsid protein of ISKNV virus particles and do not recognize MRV, which is also in the Iridoviridae family, thus exhibiting excellent specificity.

[0089] Example 4

[0090] This embodiment describes the purification, separation, and titer determination of monoclonal antibodies secreted by the selected hybridoma cell lines, specifically including the following:

[0091] (1) Obtaining monoclonal antibodies

[0092] Ten-week-old female Balb / C mice were sensitized for 10 days by intraperitoneal injection of 0.5 mL of paraffin oil. Each mouse was then injected intraperitoneally with 1 × 10⁻⁶ g of paraffin oil. 6 Hybridoma cells were collected, and mice were euthanized by cervical dislocation when their abdomens became distended and they became immobile. Ascites fluid was then collected from the abdominal cavity. The ascites fluid was centrifuged at 8000 rpm for 10 min, and the supernatant was used for affinity purification to obtain monoclonal antibodies.

[0093] The above-mentioned monoclonal antibodies were identified, and the results were consistent with expectations.

[0094] (2) Valence determination

[0095] The concentration of the monoclonal antibody obtained above was adjusted to 1 mg / mL, then serially diluted, and the antibody titer was detected by ELISA. The specific steps are as follows:

[0096] S1. Dilute the recombinant protein (i.e. the aforementioned major capsid protein) with ELISA coating buffer and coat it in a 96-well plate. Coat each well with 100 μL containing 0.1 μg of recombinant protein. Then coat overnight at 4°C and wash three times with PBST for 5 min each time.

[0097] S2. After blocking with 1% skim milk at 37°C for 1 h, wash three times with PBST for 5 min each time.

[0098] S3. Using different concentrations of the above monoclonal antibodies as primary antibodies, incubate at 37°C for 1 h, then wash three times with PBST for 5 min each time.

[0099] S4. Using HRP-conjugated Goat-anti mouse IgG (H+L) antibody as the secondary antibody, incubate at 37°C for 1 h and then wash three times with PBST for 5 min each time.

[0100] S5. Add 100 μL of single-component TMB substrate solution to each well and react for 10 min.

[0101] S6. After adding 100 μL of stop solution to each well, measure the OD. 450 When OD 450 A value greater than 0.2 is considered positive.

[0102] Test results as follows Figure 3 As shown, the titer of this monoclonal antibody is 1:320000.

[0103] The above is a detailed description of the preferred embodiments of this application, but this application is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of this application, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A monoclonal antibody against the major capsid protein of infectious spleen and kidney necrosis virus in mice, characterized in that: The monoclonal antibody comprises a heavy chain and a light chain; The heavy chain includes a heavy chain variable region, and the heavy chain variable region contains complementarity determination regions CDR-H1, CDR-H2 and CDR-H3; The amino acid sequence of the CDR-H1 is shown in SEQ ID NO.3; The amino acid sequence of the CDR-H2 is shown in SEQ ID NO.4; The amino acid sequence of the CDR-H3 is shown in SEQ ID NO.5; The light chain includes a light chain variable region, and the light chain variable region contains complementarity determination regions CDR-L1, CDR-L2 and CDR-L3; The amino acid sequence of the CDR-L1 is shown in SEQ ID NO.7; The amino acid sequence of CDR-L2 is LVS; The amino acid sequence of CDR-L3 is shown in SEQ ID NO.

8.

2. The monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

3. The monoclonal antibody according to claim 1 or 2, characterized in that: The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

6.

4. A bispecific antibody, characterized in that, It contains a monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus as described in any one of claims 1 to 3.

5. Biomaterials related to the monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus as described in any one of claims 1 to 3, characterized in that: The biomaterial comprises any one of A1) to A5); A1) A nucleic acid molecule encoding a monoclonal antibody against the major capsid protein of the mouse infectious spleen and kidney necrosis virus as described in any one of claims 1 to 3; A2) An expression cassette containing the nucleic acid molecules described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecule described in A1), the expression cassette described in A2), or the recombinant vector described in A3); A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), or the recombinant vector described in A3), wherein the recombinant cells do not contain propagation material.

6. The biomaterial according to claim 5, characterized in that: The nucleic acid molecule includes a nucleic acid molecule encoding the heavy chain variable region and a nucleic acid molecule encoding the light chain variable region.

7. A method for preparing a monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus as described in any one of claims 1 to 3, characterized in that: It is obtained by culturing the recombinant microorganisms or recombinant cells as described in claim 5 or 6, followed by isolation and purification.

8. An antibody-drug conjugate, characterized in that: Monoclonal antibodies comprising the major capsid protein of mouse anti-infective spleen and kidney necrosis virus as described in any one of claims 1 to 3.

9. A pharmaceutical composition, characterized in that: The product contains at least one of B1) to B3): B1) A monoclonal antibody against the major capsid protein of mouse infectious spleen and kidney necrosis virus as described in any one of claims 1 to 3; B2) The biomaterial as described in claim 5 or 6; B3) The antibody-drug conjugate as described in claim 8.

10. The use of the monoclonal antibody against the major capsid protein of infectious spleen and kidney necrosis virus as described in any one of claims 1 to 3 in the preparation of a reagent for detecting infectious spleen and kidney necrosis virus or its major capsid protein.

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