Monoclonal antibody specifically recognizing baculovirus gp64 protein and application thereof

Abstract

The application discloses a monoclonal antibody capable of specifically recognizing a baculovirus gp64 protein and an application thereof, the monoclonal antibody is secreted by a hybridoma cell strain 7B5-2, the hybridoma cell strain 7B5-2 has a preservation number of CGMCC No. 46131 and a preservation date of November 14, 2024. The WB experiment proves that the monoclonal antibody secreted by the hybridoma cell 7B5-2 can specifically recognize the baculovirus gp64 protein, and good application effects are achieved in an immunostaining method and a direct immunofluorescence method. The monoclonal antibody has no cross reaction with other viruses, has higher sensitivity and specificity compared with a gp64 antibody product on the market, and has higher batch precision, and can be applied to various baculovirus titer determination methods, further develops a baculovirus titer determination application scene, and provides a better antibody selection for research and development of baculovirus related diagnosis and treatment products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, and in particular to a monoclonal antibody specifically recognizing baculovirus gp64 protein and application thereof. BACKGROUND

[0002] Baculovirus expression vector system (BEVS) is a eukaryotic expression system, and the protein expressed by the system is similar to natural protein in biological activity, post-translational modification, structure and immunological activity, and can be applied in vaccines, antibodies, biological pesticides and gene therapy.

[0003] Before large-scale expression of recombinant protein, a large number of optimization tests need to be carried out to ensure the most suitable expression conditions. Among them, the inoculation amount is one of the important parameters for optimizing expression. Therefore, it is particularly important to accurately determine the titer of baculovirus.

[0004] At present, there are many methods for determining the titer of baculovirus, including plaque assay, end-point dilution, real-time quantitative PCR, flow cytometry and immunostaining. Among them, flow cytometry and immunostaining are both based on monoclonal antibodies that can specifically recognize baculovirus.

[0005] Gp64 protein is the main glycoprotein of baculovirus, which forms an envelope fusion protein with the cell membrane during viral infection, and helps the virus to fuse to the membrane of the infected cell, thereby completing the viral infection process. Therefore, the screening of monoclonal antibodies against baculovirus gp64 protein has always been of great concern.

[0006] However, the monoclonal antibodies based on gp64 available on the market are less selective, and the sensitivity and specificity need to be further improved, which seriously limits the research and development of baculovirus titer determination methods, and leads to the application of monoclonal antibodies being limited to one or two application scenarios (flow cytometry and immunostaining). SUMMARY

[0007] The purpose of the present application is to provide a monoclonal antibody that specifically recognizes baculovirus gp64 protein, which has high sensitivity, good specificity and more application scenarios.

[0008] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0009] In a first aspect, the present application provides a monoclonal antibody that specifically recognizes baculovirus gp64 protein, the heavy chain variable region amino acid sequence of which is shown in SEQ ID No. 1, and the light chain variable region amino acid sequence of which is shown in SEQ ID No. 2.

[0010] Further, the monoclonal antibody specifically recognizing the baculovirus gp64 protein is secreted by the hybridoma cell strain 7B5-2, which is preserved by the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 46131, and the preservation date is November 14, 2024.

[0011] In a second aspect, the application provides use of the monoclonal antibody specifically recognizing the baculovirus gp64 protein in preparation of a product for detecting baculovirus.

[0012] Further, the application provides use of the monoclonal antibody specifically recognizing the baculovirus gp64 protein in preparation of a product for determining the titer of baculovirus by direct immunofluorescence method.

[0013] In a third aspect, the application further provides a hybridoma cell strain 7B5-2 secreting the monoclonal antibody specifically recognizing the baculovirus gp64 protein, which is preserved by the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 46131, and the preservation date is November 14, 2024.

[0014] In a fourth aspect, the application further provides a kit for determining the titer of baculovirus, comprising the monoclonal antibody specifically recognizing the baculovirus gp64 protein.

[0015] Further, the kit for determining the titer of baculovirus is used to determine the titer of baculovirus by direct immunofluorescence method after the monoclonal antibody specifically recognizing the baculovirus gp64 protein is labeled with FITC.

[0016] Compared with the prior art, the technical effects of the application are as follows:

[0017] The application first constructs a gp64 recombinant plasmid by codon optimization, and then establishes and screens a hybridoma cell line to obtain the hybridoma cell 7B5-2 with the optimal supernatant titer and specificity, and preserve the hybridoma cell 7B5-2. Meanwhile, it is proved by WB experiment that the monoclonal antibody secreted by the hybridoma cell 7B5-2 can specifically recognize the baculovirus gp64 protein, and good application effects are achieved in immunostaining method and direct immunofluorescence method. The monoclonal antibody has no cross reaction with other viruses, has higher sensitivity and specificity than the gp64 antibody product on the market, and has higher batch precision. In summary, the monoclonal antibody specifically recognizing the baculovirus gp64 protein provided in the application has high sensitivity, good specificity and good batch precision, and can be applied to various baculovirus titer determination methods, such as direct immunofluorescence method, which further develops the application scene of baculovirus titer determination, and provides a better antibody selection for research and development of baculovirus related diagnosis and treatment products. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only represent some embodiments described in the present application, and other drawings can also be obtained by those skilled in the art based on these drawings.

[0019] Figure 1 It is a plasmid identification graph in Example 1, in which lane 1 is a plasmid double enzyme identification band, lane 2 is an original plasmid, and M lane is a nucleic acid marker.

[0020] Figure 2 It is a gp64 protein expression PAGE identification graph in Example 1, in which lane 1 is a purified gp64 protein, and M lane is a protein marker.

[0021] Figure 3 It is a gp64 protein WB identification graph in Example 1, in which lane 1 is a purified gp64 protein, lane 2 is a CHO-S cell supernatant, and M lane is a protein marker.

[0022] Figure 4 It is a mouse serum titer detection result in Example 2.

[0023] Figure 5 It is a cell supernatant ELISA detection result in Example 2.

[0024] Figure 6 It is a monoclonal antibody PAGE identification graph in Example 3, in which lane 1 is a monoclonal antibody, and M lane is a protein marker.

[0025] Figure 7 It is a monoclonal antibody WB identification graph in Example 3, in which lane 1 is a CHO-S cell supernatant, lane 2 is a gp64 protein, and M lane is a protein marker.

[0026] Figure 8 It is a FITC-labeled monoclonal antibody DFA identification graph in Example 4.

[0027] Figure 9 It is a 7B5-2 monoclonal antibody immunostaining method for detecting baculovirus content determination staining effect graph in Example 4.

[0028] Figure 10 It is a commercially available polyclonal antibody and monoclonal antibody immunostaining method for detecting baculovirus content determination staining effect graph in Example 4. DETAILED DESCRIPTION

[0029] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores.

[0030] Example 1: Preparation of Immunogen

[0031] 1.1 Construction of gp64 recombinant plasmid

[0032] The full-length sequence of the baculovirus gp64 gene (NC_001623) downloaded from GenBank was used to predict the extracellular region and signal peptide of gp64 using TMHMM and SignalP-4.1 online software. The results showed that amino acids 1-20 constituted the signal peptide, and amino acids 21-482 constituted the extracellular region. To obtain a soluble protein, the extracellular region of the gp64 protein was selected for expression. Specifically, the sequence of amino acids 21-482 of the gp64 gene was optimized according to CHO cell codon preference, and the IL-2 signal peptide sequence ATGTACAGAATGCAGCTGCTGTCTTGTATCGCCCTGTCTCTGGCCCTGGTGACCAACAGC was added to the 5' end. A six-histidine tag CACCACCATCACCACCAC was added before the 3' stop codon. This was synthesized by GenScript Biotech Co., Ltd., ligated into the pcDNA3.4 vector via the Xba I + EcoR V restriction site, and named pcDNA-gp64. Plasmid identification is as follows. Figure 1 As shown, lane 1 is a double enzyme digestion map, which contains the vector fragment (approximately 6011 bp) and the target fragment (approximately 1500 bp), both of which are consistent with the expected size. Lane 2 is the original plasmid, and lanes M1 and M2 are markers.

[0033] 1.2 Expression and identification of gp64 protein

[0034] The prepared pcDNA3.4 plasmid containing the target sequence was transformed and cultured in a culture medium. The plasmid was then extracted using an endotoxin-free plasmid extraction kit. The extracted plasmid was transiently transfected into CHO-S cells, and after 10–12 days of culture, the cell culture supernatant was harvested. The gp64 protein was purified using Ni column affinity chromatography, and the expressed gp64 protein was identified by PAGE and Western blotting. PAGE identification results are as follows: Figure 2As shown, lane 1 contains the purified gp64 protein, with a size between 50KD and 70KD, and lane M is the marker. Western blotting identification is as follows. Figure 3 As shown, lane 1 contains purified gp64 protein, lane 2 contains CHO cell supernatant, and lane M contains marker. Western blotting was performed using His-Tag monoclonal antibody as the primary antibody.

[0035] Example 2: Establishment and screening of hybridoma cell lines

[0036] 2.1 Mouse Immunization

[0037] Five female Balb / c mice were immunized with purified and identified gp64 protein, labeled as 1#, 2#, 3#, 4#, and 5#. The initial immunization dose was 60 μg / mouse, using Freund's complete adjuvant. Three booster immunizations were administered, each with 30 μg / mouse, using Freund's incomplete adjuvant. The final immunization was 50 μg / mouse. Serum titers after the four immunizations were detected using an indirect ELISA method. The indirect ELISA method used purified gp64 protein coated at 0.5 μg / well. The sample consisted of serum from five mice. The secondary antibody was goat anti-mouse HRP antibody, and the substrate was TMB. The ELISA titers of the mouse serum after the four immunizations are shown below. Figure 4 As shown, therefore, mice #3 with high positive screening titer and negative negative screening were selected for final immunization, and cell fusion was performed 4 days after final immunization.

[0038] 2.2 Cell fusion and ELISA screening

[0039] Immunosplenic cells were mixed with SP2 / 0 at a ratio of 6:1 in a sterile centrifuge tube. The cells were washed with 1640 incomplete culture medium by centrifugation, and residual liquid was aspirated. The bottom of the centrifuge tube was gently tapped to create gaps between the cells in the pellet. While preheating the tube in a 37°C water bath, 0.8 ml of preheated 50% PEG4000 (37°C) was pipetted into the centrifuge tube over 60 seconds, gently shaking the tube during the addition. The preheated 1640 incomplete culture medium (37°C) was then added using a sterile pipette at a rate decreasing from fast to slow. The fusion tube was then incubated at 37°C for 25 minutes. The cells were centrifuged, the supernatant was discarded, and 1640 complete culture medium containing HAT was added to resuspend the fused cell pellet. A 96-well plate pre-coated with feeder cells was taken out, and 100 µl of the fused cell suspension was added to each well, which was then labeled. The 96-well plates were placed in a cell culture incubator. When cell fusion clusters appeared and a sufficient number of fused cells were obtained, the wells were labeled, and the fusion wells were screened using an indirect ELISA method. Fourteen positive hybridoma parent clones were selected, and the ELISA results are shown in Table 1.

[0040] Table 1. Results of ELISA detection of maternal clone cell supernatant

[0041]

[0042] Cloning of positive wells was performed. Feeder cells were prepared the day before cloning. Cells from fusion wells showing positive results were aspirated into 15 ml sterile centrifuge tubes containing 1 ml of cell culture medium using a pipette and labeled. Cells from each well were counted. Based on the cell count, cells from each well were diluted to three different dilutions: 5–10 cells / ml, 10–20 cells / ml, and 20–30 cells / ml. Cell suspensions at different dilutions were added to 96-well plates containing feeder cells, 100 µl / well, and labeled. The 96-well plates were incubated in a cell culture incubator. When cell clusters appeared and a sufficient number of fusion cells were obtained, they were labeled, and the fusion wells were screened using an indirect ELISA method. Ten positive hybridoma cell lines were screened after cloning. The titers of the cell supernatant are shown in [link to cell culture results]. Figure 5 As can be seen from the figure, the ELISA titers of 13E1-2, 4B2-2, 20A5-2, and 25F9-1 are relatively low, while the ELISA titers of 2B7-1, 4H6-1, 5A3-1, 7B5-2, 11A10-2, and 23A8-1 are relatively high.

[0043] 2.3 Screening by Immunostaining

[0044] Immunostaining was performed on the supernatants of six monoclonal cell lines with high ELISA titers (2B7-1, 4H6-1, 5A3-1, 7B5-2, 11A10-2, and 23A8-1). Inoculation wells and cell control wells were included, and the detailed experimental procedures were as described in section 4.3. The cell supernatants were not diluted. The staining results showed that all six monoclonal cell lines stained blue in the inoculation wells, but the cell control wells showed significant differences. Specifically, the 11A10-2 and 23A8-1 cell control wells contained a large number of single blue cells. The blue spots of the other four clones showed good morphology, and the cell control wells contained virtually no non-specific blue cells.

[0045] 2.4 Indirect immunofluorescence screening

[0046] Indirect immunofluorescence identification was performed on the supernatants of four monoclonal cell lines screened by immunostaining. Inoculation wells and cell control wells were set up, and the experimental procedure is described in 4.2.2. In 4.2.2, the cell supernatants were incubated instead of the monoclonal antibody, followed by incubation with FITC-labeled goat anti-mouse secondary antibody. The staining results showed no fluorescence in the supernatants of all four cell lines and the cell control wells, while the fluorescence in the inoculation wells was relatively bright, indicating that the immunofluorescence assay was successful. The best-performing cell line, 7B5-2, was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The accession number is CGMCC No. 46131, and the deposit date is November 14, 2024. Classification and nomenclature: Baculovirus AcMNPV hybridoma cell line. Mus musculus .

[0047] Example 3: Preparation and Identification of Monoclonal Antibodies

[0048] 3.1 Monoclonal Antibody Preparation

[0049] Female Balb / c mice aged 8–10 weeks were selected and injected intraperitoneally with 500 μl of sterile liquid paraffin per mouse. Seven days later, each mouse was injected with 500 μl of 7B5-2 cell suspension, with a cell density of 2–4 × 10⁻⁶ cells. 6 Cells / ml. About 7 days after cell injection, significant distension of the peritoneum in mice can be observed. Ascites fluid can be collected using a 20-gauge needle, and multiple collections are possible.

[0050] The collected ascites fluid was centrifuged to remove impurities, then purified using a Protein G column. The column was equilibrated with 20 mM PBS (pH 7.2), eluted with Gly-HCl solution (pH 2.7), and neutralized with Tris-HCl (pH 9.0). The resulting monoclonal antibody was then subjected to G25 column chromatography with the buffer replaced by PBS solution at pH 7.2. The obtained monoclonal antibody was aliquoted and stored at -20°C.

[0051] 3.2 Identification of Monoclonal Antibodies

[0052] The monoclonal antibody (hereinafter referred to as 7B5-2 monoclonal antibody or 7B5-2 monoclonal antibody) was identified by reduction SDS-PAGE, and the results are as follows: Figure 6 As shown, the purity of the 7B5-2 monoclonal antibody is 93.16%. The 7B5-2 monoclonal antibody was sequenced, and the amino acid sequence of the heavy chain variable region of the 7B5-2 monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the 7B5-2 monoclonal antibody is shown in SEQ ID NO: 2.

[0053] SEQ ID NO: 1

[0054] EVQLQESGPSLVKPSQTLSLACSVTGDSITSGYWNWIRKFPGNKLEYMGFISYSGNTYYNPSLRSRISITRDTSKNQYYLQLNSVTTEDTATYYCARGYANPEDFDSWGQGTTLTVSS.

[0055] SEQ ID NO: 2

[0056] IEFACRPFLLLWFPGTCDIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQFAGYPLTFGAGTKLELK.

[0057] Example 4: Application of Monoclonal Antibodies

[0058] 4.1 Application of monoclonal antibodies in Western blotting experiments

[0059] The purified gp64 protein was subjected to reducing SDS-PAGE, with sf9 cell supernatant as the negative control. Separation was performed using 10% SDS-PAGE, followed by transfer to an NC membrane at a constant current of 300 mA. The membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight with 7B5-2 monoclonal antibody (1:1000 dilution). After washing five times with PBST, HRP-labeled goat anti-mouse secondary antibody was added, and the membrane was incubated at room temperature for 1 h. Then, the prepared ECL chemiluminescent substrate was added to the NC membrane, and the membrane was exposed, developed, and photographed in a fully automated chemiluminescence system. The results were then saved. Figure 7 As can be seen after exposure, there is only one band, with a size between 50KD and 70KD. This indicates that the screened 7B5-2 monoclonal antibody can specifically bind to the gp64 protein.

[0060] 4.2 FITC-labeled monoclonal antibody and direct immunofluorescence identification (DFA)

[0061] 4.2.1 FITC labeling of monoclonal antibodies

[0062] The purified 7B5-2 monoclonal antibody was replaced with a G25 chromatography column using a carbonate buffer system at pH 9.3. A 1 mg / ml FITC-DMSO solution was prepared, and 50 μl of FITC-DMSO solution was added per milliliter of protein solution for labeling. The protein solution was placed on a magnetic stirrer, and FITC-DMSO solution was added dropwise while stirring. Labeling was performed at room temperature for 4 h, protected from light throughout. After labeling, the buffer system was replaced with a G25 chromatography column using a 10 mM PBS solution at pH 7.2, and the antibody concentration was adjusted to 1 mg / ml.

[0063] 4.2.2 Direct immunofluorescence assay

[0064] (1) Plating and inoculation: Sf9 cells in the logarithmic growth phase were plated and, after the cell monolayer was formed, recombinant baculovirus expressing canine parvovirus VP2 protein was inoculated and cultured at 27°C for 48 h.

[0065] (2) Fixation: Discard the liquid and fix with 70% ethanol for 30 min, 200 μl / well;

[0066] (3) Washing: Wash the plate 3 times with PBST, 200 μl / well;

[0067] (4) Antibody incubation: The labeled monoclonal antibody was diluted at 1:100, 1:300 and 1:500, and 50 µl was incubated in each well at 37°C for 30 min.

[0068] (5) Washing: Wash the plate 3 times with PBST, 200 μl / well;

[0069] (6) Observation under a fluorescence microscope, results Figure 8 As shown.

[0070] from Figure 8 The results showed that the labeled 7B5-2-FITC monoclonal antibody was effective in direct immunofluorescence detection at all three dilutions for cell controls, with very bright fluorescence. This indicates that the prepared 7B5-2 monoclonal antibody, after FITC labeling, exhibits excellent direct immunofluorescence detection performance, meeting the application requirements. This demonstrates that the antibody can be used in Reed-Muench calculations of baculovirus viral titers, where the titer represents the median tissue cell infection dose. Furthermore, meeting this application requirement indicates that the 7B5-2 monoclonal antibody can be used for the specificity testing of baculovirus biological products.

[0071] In addition, F81 cell samples infected with feline panleukopenia virus, feline calicivirus, and feline herpesvirus, MDBK cell samples infected with bovine viral diarrhea virus, and Vero cell samples infected with canine distemper virus were prepared and directly identified by immunofluorescence using the 7B5-2-FITC antibody according to the experimental procedure. The results showed that only the recombinant baculovirus inoculation wells showed fluorescence, while the inoculation wells of other samples and the cell control wells showed no fluorescence, indicating that the screened 7B5-2 monoclonal antibody has good specificity, can specifically recognize baculoviruses, and has no cross-reactivity with other viruses.

[0072] 4.3 Detection of baculovirus content by immunostaining

[0073] (1) Cell plating: Sf9 cells in the logarithmic growth phase were dispersed, counted, and inoculated into 96-well cell culture plates at a density of 6.5 × 10⁶ cells per well. 4One cell, cultured at 27°C for 1 hour;

[0074] (2) Virus dilution: The baculovirus to be tested was serially diluted 10-fold using serum-free insect cell culture medium. Take 10 -3 10 -4 10 -5 There are 3 dilution levels in total;

[0075] (3) Virus inoculation: Take a 96-well cell culture plate that has been cultured at 27°C for 1 hour, aspirate the cell supernatant, add 25 µl of virus solution of various dilutions to each well, and 10 -3 10 -4 10 -5 Inoculate 4 wells with each dilution, leaving 1 well as a negative control, and add 25 µl of culture medium. Gently shake the 96-well cell culture plate to distribute the virus evenly. Incubate at 27°C in a humidified chamber for 1 hour to allow the virus to absorb. Aspirate the inoculated virus solution and add 50 µl of self-prepared 0.5% methylcellulose to each well.

[0076] (4) Culture: Place the 96-well cell culture plate in an incubator and culture at 27°C for 43-47 hours;

[0077] (5) Cell fixation: Slowly add 150 µl of 80% acetone to each well and fix at room temperature for 30 minutes;

[0078] (6) Washing: Discard the acetone, pat gently on a paper towel, and wash 3 times with PBST, 200 μl per well;

[0079] (7) Blocking: Add 50 μl of 30-fold diluted goat serum to each well for blocking, and incubate gently at room temperature for 5 minutes;

[0080] (8) Add primary antibody: Discard the blocking solution, pat gently on a paper towel, add 50 µl of 400-fold diluted 7B5-2 monoclonal antibody to each well, and incubate at 37°C for 25 minutes. The concentration of 7B5-2 monoclonal antibody is 0.554 mg / ml;

[0081] (9) Washing: Discard the primary antibody, pat gently on a paper towel, and wash twice with PBST, 200 μl per well, 5 minutes each time;

[0082] (10) Add secondary antibody: Add 50 µl of HRP-labeled goat anti-mouse antibody working solution diluted to 250-fold to each well and incubate at 37°C for 25 minutes.

[0083] (11) Washing: Discard the secondary antibody, pat gently on a paper towel, and wash 3 times with PBST, 200 μl per well, 5 minutes each time;

[0084] (12) Color development: Add 50 μl of peroxidase substrate to each well and react at room temperature in the dark for 1-3 hours;

[0085] (13) Microscopic observation: Under an optical microscope, no blue spots appeared in the normal cell control wells, indicating that the experiment was successful; count the number of blue spots in the wells of the samples to be tested;

[0086] (14) Virus content calculation: Virus content (IFU / ml) = average number of blue spots × reciprocal of dilution × 40.

[0087] Figure 9 The images show the state of the cell control and sample wells after staining. The cell control wells show no non-specific blue spots, while the inoculation wells show clear and well-formed blue spots. This result indicates that the screened 7B5-2 monoclonal antibody can be applied to the determination of baculovirus titers using immunostaining.

[0088] Simultaneously, this invention also uses a commercially available polyclonal antibody against gp64 protein and a gp64 monoclonal antibody to perform immunostaining to determine the staining status of baculovirus titers according to the experimental procedures described above. The results are as follows... Figure 10 The results showed that the blue spots of the gp64 polyclonal antibody inoculation wells were distinguishable but irregular, while the cell control showed a large number of single or small clusters of blue cells; the blue spots of the gp64 monoclonal antibody were poorly shaped and difficult to distinguish, and the cell control appeared entirely blue. Therefore, the 7B5-2 monoclonal antibody provided by this invention is significantly superior to commercially available gp64 polyclonal antibody and monoclonal antibody products in the application of immunostaining for the determination of baculovirus titers.

[0089] Furthermore, following the experimental steps described above, the amount of antibody incubated for 7B5-2 monoclonal antibody staining was small (only 0.069 μg / well), and the sensitivity was high. Moreover, the results of three titer determinations of the same batch of baculovirus showed that the 7B5-2 monoclonal antibody had good inter-batch precision in the immunostaining method for determining baculovirus titers, with a viral load CV of 12.84% (less than 30%) in the three determinations, as shown in Table 2.

[0090] Table 2 Results of three assays for the same batch of baculovirus

[0091]

[0092] The above description of the disclosed embodiments is intended to enable those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of the claims of this invention.

Claims

1. A monoclonal antibody that specifically recognizes the gp64 protein of baculovirus, characterized in that, Its heavy chain variable region amino acid sequence is shown in SEQ ID No.

1. Its light chain variable region amino acid sequence is shown in SEQ ID No.

2.

2. The monoclonal antibody specifically recognizing baculovirus gp64 protein according to claim 1, characterized in that, It is secreted by hybridoma cell line 7B5-2, which is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46131 and deposit date of November 14, 2024.

3. The use of the monoclonal antibody that specifically recognizes the gp64 protein of baculovirus according to claim 1 in the preparation of products for detecting baculovirus.

4. The application of the monoclonal antibody that specifically recognizes the baculovirus gp64 protein according to claim 1 in the preparation of products for direct immunofluorescence assay of baculovirus titers.

5. A hybridoma cell line 7B5-2 that secretes the monoclonal antibody as described in claim 1, which is deposited by the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46131 and deposit date of November 14, 2024.

6. A kit for determining baculovirus titers, characterized in that, A monoclonal antibody comprising the specific recognition of the baculovirus gp64 protein as described in claim 1.

7. The kit for determining baculovirus titers according to claim 6, characterized in that, The monoclonal antibody that specifically recognizes the baculovirus gp64 protein was labeled with FITC, and the baculovirus titer was determined by direct immunofluorescence.

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