Monoclonal antibody for specifically recognizing baculovirus gp64 protein and application thereof

Abstract

The invention discloses a monoclonal antibody for specifically recognizing baculovirus gp64 protein and application of the monoclonal antibody, the monoclonal antibody is secreted by a hybridoma cell strain 7B5-2, the preservation number of the hybridoma cell strain 7B5-2 is CGMCC (China General Microbiological Culture Collection Center) No.46131, and the preservation date is November 25, 2024. WB experiments prove that the monoclonal antibody secreted by the hybridoma cell 7B5-2 can specifically recognize baculovirus gp64 protein, achieves a good application effect in an immunostaining method and a direct immunofluorescence method, does not have cross reaction with other viruses, and has higher sensitivity and specificity and higher inter-batch precision compared with a gp64 antibody product in the market, so that the monoclonal antibody can be used for detecting the baculovirus gp64 protein. The method can be applied to various baculovirus titer determination methods, the application scene of baculovirus titer determination is further expanded, and a better antibody choice is provided for research and development of baculovirus related diagnosis and treatment products.

Description

Technical Field

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

[0002] The baculovirus expression vector system (BEVS) is a eukaryotic expression system. The proteins expressed by this system are similar to natural proteins in terms of biological activity, post-translational modification, structure and immunological activity, and can be used in vaccines, antibodies, biopesticides and gene therapy.

[0003] Before large-scale expression of recombinant proteins, a large number of optimization experiments are required to ensure the most appropriate expression conditions. Among them, the inoculum size is one of the important parameters for optimizing expression. Therefore, it is particularly important to accurately determine the titer of baculovirus.

[0004] There are many methods for measuring baculovirus titer, including plaque assay, endpoint dilution method, real-time quantitative PCR, flow cytometry and immunostaining. Among them, flow cytometry and immunostaining are based on monoclonal antibodies that can specifically recognize baculovirus.

[0005] The gp64 protein is the main glycoprotein of baculovirus. During viral infection, it fuses with the cell membrane to form an envelope fusion protein, which helps the virus fuse to the infected cell membrane and complete the viral infection process. Therefore, the screening of monoclonal antibodies against the baculovirus gp64 protein has always attracted much attention.

[0006] However, there are currently few gp64-based monoclonal antibodies on the market, and their sensitivity and specificity need to be further improved. This situation severely limits the research and development of baculovirus titer determination methods, resulting in the application of monoclonal antibodies being limited to one or two application scenarios (flow cytometry and immunostaining). Summary of the invention

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

[0008] In order to achieve the above object, the technical solution of the present invention is:

[0009] In a first aspect, the present invention provides a monoclonal antibody that specifically recognizes baculovirus gp64 protein, wherein the amino acid sequence of the heavy chain variable region 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.

[0010] Furthermore, the above-mentioned monoclonal antibody that specifically recognizes baculovirus gp64 protein is secreted by the hybridoma cell line 7B5-2, and the hybridoma cell line 7B5-2 is deposited with the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No.46131 and the deposit date of November 14, 2024.

[0011] In a second aspect, the present invention provides the use of the above-mentioned monoclonal antibody that specifically recognizes baculovirus gp64 protein in the preparation of a product for detecting baculovirus.

[0012] Furthermore, the present invention provides the use of the above-mentioned monoclonal antibody that specifically recognizes baculovirus gp64 protein in the preparation of a product for determining the titer of baculovirus by direct immunofluorescence method.

[0013] In the third aspect, the present invention also provides a hybridoma cell line 7B5-2 that secretes a monoclonal antibody that specifically recognizes baculovirus gp64 protein, which is deposited by the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.46131 and a deposit date of November 14, 2024.

[0014] In a fourth aspect, the present invention further provides a kit for determining the titer of baculovirus, comprising the above-mentioned monoclonal antibody that specifically recognizes baculovirus gp64 protein.

[0015] Furthermore, in the above-mentioned kit for determining the baculovirus titer, the monoclonal antibody that specifically recognizes the baculovirus gp64 protein is labeled with FITC, and the baculovirus titer is determined by direct immunofluorescence method.

[0016] Compared with the prior art, the technical effects of the present invention are:

[0017] The present invention first constructs a gp64 recombinant plasmid by codon optimization, and then obtains the hybridoma cell 7B5-2 with the best cell supernatant titer and specificity after the establishment and screening of the hybridoma cell line, and preserves it. At the same time, WB experiments have proved that the monoclonal antibody secreted by the hybridoma cell 7B5-2 can specifically recognize the baculovirus gp64 protein, and has achieved good application results in both immunostaining and direct immunofluorescence, without cross-reaction with other viruses, and has higher sensitivity and specificity than the gp64 antibody products on the market, and has higher batch precision. In summary, the monoclonal antibody that specifically recognizes the baculovirus gp64 protein provided by the present application has high sensitivity, good specificity, and good batch precision. It can be applied to a variety of baculovirus titer determination methods, such as direct immunofluorescence, and further expands the application scenario of baculovirus titer determination, providing a better antibody selection for the research and development of baculovirus-related diagnosis and treatment products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can also be obtained based on these drawings.

[0019] Figure 1 This is a plasmid identification diagram in Example 1, wherein lane 1 is a plasmid double enzyme digestion identification band, lane 2 is the original plasmid, and lane M is a nucleic acid marker.

[0020] Figure 2 This is a PAGE identification diagram of the gp64 protein expressed in Example 1, wherein lane 1 is the purified gp64 protein and lane M is the protein marker.

[0021] Figure 3 This is a WB identification diagram of the gp64 protein expressed in Example 1, wherein lane 1 is the purified gp64 protein, lane 2 is the CHO-S cell supernatant, and lane M is the protein marker.

[0022] Figure 4 This is the mouse serum titer test result in Example 2.

[0023] Figure 5 This is the ELISA test result of the cell supernatant in Example 2.

[0024] Figure 6 This is the PAGE identification diagram of the monoclonal antibody in Example 3, wherein lane 1 is the monoclonal antibody and lane M is the protein marker.

[0025] Figure 7 This is a WB identification diagram of the monoclonal antibody in Example 3, wherein lane 1 is the supernatant of CHO-S cells, lane 2 is the gp64 protein, and lane M is the protein marker.

[0026] Figure 8 This is the identification diagram of the FITC-labeled monoclonal antibody DFA in Example 4.

[0027] Fig. 9 This is a staining effect diagram of the 7B5-2 monoclonal antibody immunostaining method for detecting baculovirus content in Example 4.

[0028] Fig.10 This is a staining effect diagram of the commercially available polyclonal antibody and monoclonal antibody immunostaining method for detecting baculovirus content in Example 4. DETAILED DESCRIPTION

[0029] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods in the following embodiments are conventional methods unless otherwise specified. The test materials used in the following embodiments are purchased from conventional biochemical reagent stores unless otherwise specified.

[0030] Example 1 Preparation of immunogen

[0031] 1.1 Construction of gp64 recombinant plasmid

[0032] The full-length sequence of the baculovirus gp64 gene downloaded from GenBank (NC_001623) was used to predict the extracellular region and signal peptide of gp64 using TMHMM and SignalP-4.1 online software. The results showed that the 1st to 20th amino acids were signal peptides, and the 21st to 482nd amino acids were the extracellular region. In order to obtain soluble protein, the extracellular region of gp64 protein was selected for expression, that is, the 21st to 482nd amino acid sequence of the gp64 gene was optimized according to the codon preference of CHO cells, and the IL-2 signal peptide sequence was added to the 5' end.

[0033] ATGTACAGAATGCAGCTGCTGTCTTGTATCGCCCTGTCTCTGGCCCTG GTGACCAACAGC, a 6-histidine tag CACCACCATCACCACCAC was inserted before the 3' terminator codon, synthesized by GenScript Biotech Co., Ltd., and ligated into the pcDNA3.4 vector through the Xba I + EcoR V restriction site, named pcDNA-gp64. Plasmid identification is as follows Figure 1 As shown, lane 1 is a double enzyme digestion map, which is a vector fragment (about 6011 bp) and a target fragment (about 1500 bp), both of which are consistent with the expected size, lane 2 is the original plasmid, and lanes M1 and M2 are markers.

[0034] 1.2 Expression and identification of gp64 protein

[0035] After the prepared pcDNA3.4 plasmid containing the target sequence was transformed and cultured, the plasmid was extracted using an endotoxin-free plasmid extraction kit, and the extracted plasmid was transiently transfected into CHO-S cells. After culturing for 10 to 12 days, the cell culture supernatant was harvested. The gp64 protein was affinity purified using a Ni column, and the expressed gp64 protein was identified by PAGE and WB. PAGE identification was as follows Figure 2As shown, lane 1 is the purified gp64 protein, with a size between 50KD and 70KD, and lane M is the marker. Figure 3 As shown, lane 1 is the purified gp64 protein, lane 2 is the CHO cell supernatant, and lane M is the marker. The primary antibody used for WB identification is His-Tag monoclonal antibody.

[0036] Example 2 Establishment and screening of hybridoma cell lines

[0037] 2.1 Immunization of mice

[0038] Five Balb / c female mice were immunized with the purified and identified gp64 protein, and they were marked as 1#, 2#, 3#, 4#, and 5#. The first immunization dose was 60 μg / mouse, and the adjuvant was Freund's complete adjuvant; three booster immunizations were performed, each time at 30 μg / mouse, and the adjuvant was Freund's incomplete adjuvant; the final immunization was 50 μg / mouse. The indirect ELISA method was used to detect the titer of mouse serum after four immunizations. The indirect ELISA method was coated with purified gp64 protein, 0.5 μg / well, and the samples to be tested were sera from 5 mice. The secondary antibody was goat anti-mouse-HRP antibody, and the substrate was TMB. The ELISA titer of mouse serum after four immunizations is as follows Figure 4 As shown, therefore, 3# mice with high positive screening titer and negative reverse screening were selected for final immunization, and cell fusion was performed 4 days after the final immunization.

[0039] 2.2 Cell fusion and ELISA screening

[0040] Mix immune spleen cells and SP2 / 0 in a sterile centrifuge tube at a ratio of 6:1, wash the cells with 1640 incomplete culture medium, and aspirate the residual liquid. Gently tap the bottom of the centrifuge tube to create gaps between the precipitated cells. While preheating in a 37°C water bath, use a 1ml pipette to aspirate 0.8ml of 50% PEG4000 preheated at 37°C. Add it dropwise to the centrifuge tube within 60 seconds, and gently shake the centrifuge tube during the addition process. After adding 1640 incomplete culture medium preheated at 37°C with a sterile pipette at a speed from fast to slow, place the fusion tube in a 37°C incubator for 25 minutes. Centrifuge the cells, discard the supernatant, add 1640 complete culture medium containing HAT, and suspend the fused cell pellet. Take out the 96-well plate with feeder cells in advance, add 100μl / well of the fusion cell suspension, and mark it. The 96-well plate was placed in a cell culture incubator for culture. When cell fusion clusters appeared and the number of fusion cells reached a sufficient level, they were marked and the fusion wells were screened using the indirect ELISA method. 14 positive hybridoma mother clones were screened, and the ELISA results are shown in Table 1.

[0041] Table 1 ELISA test results of parent clone cell supernatant

[0042]

[0043]

[0044] The positive wells were cloned. The feeder cells were prepared one day before the cloning of the positive wells. The cells of the fusion wells with positive test results were aspirated into a 15ml sterile centrifuge tube containing 1ml of cell culture medium with a pipette and marked. Count the cells aspirated from each well. According to the cell count, the cells in each well were diluted to three different dilutions of 5-10 / ml, 10-20 / ml, and 20-30 / ml. The cell suspensions of different dilutions of the cells in each well were added to a 96-well plate with feeder cells, 100μl / well, and marked. The 96-well plate was cultured in a cell culture incubator. When cell clusters appeared and the fusion cells reached a sufficient amount, they were marked and the fusion wells were screened by indirect ELISA. After cloning, a total of 10 positive hybridoma cell lines were screened, and the titer of the cell supernatant was detected as shown in the figure. 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.

[0045] 2.3 Immunostaining screening

[0046] The supernatants of 6 monoclonal cells with high ELISA titers (2B7-1, 4H6-1, 5A3-1, 7B5-2, 11A10-2 and 23A8-1) were stained by immunostaining, with virus-infected wells and cell control wells. The detailed experimental steps were the same as those shown in 4.3, and the cell supernatants were not diluted. The staining results showed that the virus-infected wells of the 6 monoclonal supernatants all stained blue spots, but the cell controls were quite different. Among them, the 11A10-2 and 23A8-1 cell control wells had more single blue cells. The blue spot morphology of the other 4 clones was better, and there were basically no non-specific blue cells in the cell controls.

[0047] 2.4 Indirect immunofluorescence screening

[0048] The supernatants of the four monoclonal cells screened by the immunostaining method were subjected to indirect immunofluorescence identification. The virus-infected wells and cell control wells were set up. The experimental steps are shown in 4.2.2. When incubating the monoclonal antibody in 4.2.2, the cell supernatant was incubated instead, and then the FITC-labeled goat anti-mouse secondary antibody was incubated. The staining results showed that the supernatants of the four cell strains and the cell control wells had no fluorescence, and the fluorescence of the virus-infected wells was relatively bright, and the immunofluorescence experiments were all established. The best 7B5-2 was selected for preservation. The preservation institution is the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The preservation number is CGMCC No.46131, and the preservation date is November 14, 2024. Classification name: Baculovirus AcMNPV hybridoma cell line Mus musculus.

[0049] Example 3 Preparation and identification of monoclonal antibodies

[0050] 3.1 Monoclonal Antibody Preparation

[0051] Female Balb / c mice aged 8 to 10 weeks were selected and sterilized liquid paraffin was injected intraperitoneally, 500 μl per mouse. Seven days later, each mouse was injected with 500 μl of 7B5-2 cell suspension at a cell density of 2 to 4 × 10 6 About 7 days after the cell injection, the abdominal cavity of the mouse was obviously enlarged, and the ascites was collected with a 20-gauge needle. The collection can be repeated multiple times.

[0052] After the collected ascites was centrifuged and impurities removed, it was purified using a Protein G column, equilibrated with 20mM PBS at pH 7.2, eluted with a Gly-HCl solution at pH 2.7, neutralized with Tris-HCl at pH 9.0, and the obtained monoclonal antibody was replaced with a PBS solution at pH 7.2 by G25 column chromatography. The obtained monoclonal antibody was aliquoted and stored at -20°C.

[0053] 3.2 Identification of monoclonal antibodies

[0054] The monoclonal antibody (hereinafter referred to as 7B5-2 monoclonal antibody or 7B5-2 monoclonal antibody) was identified by reducing SDS-PAGE. The results are as follows Figure 6 As shown, the purity of 7B5-2 monoclonal antibody is 93.16%. 7B5-2 monoclonal antibody was sequenced, and the amino acid sequence of the heavy chain variable region of 7B5-2 monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of 7B5-2 monoclonal antibody is shown in SEQ ID NO:2.

[0055] SEQ ID NO:1

[0056] EVQLQESGPSLVKPSQTLSLACSVTGDSITSGYWNWIRKFPGNKLEY MGFISYSGNTYYNPSLRSRISITRDTSKNQYYLQLNSVTTEDTATYYCAR GYANPEDFDSWGQGTTLTVSS.

[0057] SEQ ID NO:2

[0058] IEFACRPFLLLWFPGTCDIQMTQSPSSLSASLGERVSLTCRASQEIS GYLSWLQQKPDGTIKRLIYAASTLDSGVPKRFSGSRSGSDYSLTISSLESE DFADYYCLQFAGYPLTFGAGTKLELK.

[0059] Example 4 Application of monoclonal antibodies

[0060] 4.1 Application of monoclonal antibodies in WB experiments

[0061] The purified gp64 protein was subjected to reducing SDS-PAGE, and the negative control was the supernatant of sf9 cells. It was separated by 10% SDS-PAGE, transferred to the NC membrane at 300mA constant current, blocked with 5% skim milk powder at room temperature for 1 hour, and incubated with 7B5-2 monoclonal antibody (1:1000 dilution) overnight. Washed with PBST five times, HRP-labeled goat anti-mouse secondary antibody was added, incubated at room temperature for 1 hour, and the prepared ECL chemiluminescent substrate was added to the NC membrane, exposed, developed, photographed, and the results were saved in the fully automatic chemiluminescence system. Figure 7 It can be seen that there is only one band after exposure, with a size between 50KD and 70KD, indicating that the screened 7B5-2 monoclonal antibody can specifically bind to gp64 protein.

[0062] 4.2 FITC-labeled monoclonal antibodies and direct immunofluorescence assay (DFA)

[0063] 4.2.1 FITC labeling of monoclonal antibodies

[0064] The purified 7B5-2 monoclonal antibody was replaced with a carbonate system with a pH of 9.3 using a G25 chromatography column, and a 1mg / ml FITC-DMSO solution was prepared. 50μl of FITC-DMSO solution was added per milliliter of protein solution for labeling. The protein solution was placed on a magnetic stirrer, and the FITC-DMSO solution was added dropwise while stirring. The labeling was performed at room temperature for 4h, and the whole process was protected from light. After the labeling was completed, the buffer system was replaced with a PBS solution with a pH of 7.210mM ​​using a G25 chromatography column, and the antibody concentration was adjusted to 1mg / ml.

[0065] 4.2.2 Direct immunofluorescence test

[0066] (1) Plating and inoculation: Sf9 cells in the logarithmic growth phase were plated and inoculated with recombinant baculovirus expressing canine parvovirus VP2 protein after the cells became monolayers. The cells were cultured at 27°C for 48 h.

[0067] (2) Fixation: discard the solution and fix with 70% ethanol for 30 min, 200 μl / well;

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

[0069] (4) Antibody incubation: dilute the labeled monoclonal antibody at 1:100, 1:300, and 1:500, incubate 50 μl per well, and incubate at 37°C for 30 min;

[0070] (5) Washing: Wash the plate three times with PBST, 200 μl / well;

[0071] (6) Fluorescence microscopy observation, results Figure 8 shown.

[0072] from Figure 8 The results show that the labeled 7B5-2-FITC monoclonal antibody has three dilution multiples of cell control in direct immunofluorescence detection, and the fluorescence is very bright. This shows that the prepared 7B5-2 monoclonal antibody has a good effect in direct immunofluorescence detection after being labeled with FITC, which meets the application function, indicating that the antibody can be used for Reed-Muench calculation of baculovirus virus titer, and the titer indicates the result as the infection amount of half tissue cells. In addition, it meets this application, indicating that 7B5-2 monoclonal antibody can be used for specific testing of baculovirus biological products.

[0073] 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 direct immunofluorescence identification was performed using 7B5-2-FITC antibody according to the experimental procedures. The results showed that only the recombinant baculovirus-infected wells had fluorescence, while the other sample-infected wells and cell control wells had no fluorescence, indicating that the screened 7B5-2 monoclonal antibody has good specificity, can specifically identify baculovirus, and has no cross-reaction with other viruses.

[0074] 4.3 Immunostaining to detect baculovirus content

[0075] (1) Cell plating: Sf9 cells in the logarithmic growth phase were dispersed, counted, and plated into a 96-well cell culture plate, with 6.5×10 cells per well. 4 cells, cultured at 27°C for 1 hour;

[0076] (2) Virus dilution: The baculovirus to be tested was serially diluted 10-fold using insect cell serum-free culture medium. -3 , 10 -4 , 10 -5 There are 3 dilutions in total;

[0077] (3) Virus inoculation: Take a 96-well cell culture plate that has been cultured at 27°C for 1 hour, aspirate the cell supernatant, and add 25 μl of each diluted virus solution to each well. -3 , 10 -4 , 10 -5 Inoculate 4 wells at each dilution, leave 1 well as a negative control, and add 25 μl of culture medium. Gently shake the 96-well cell culture plate to evenly distribute the virus. Place in a 27°C wet box for 1 hour, aspirate the inoculated virus solution, and add 50 μl of self-prepared 0.5% methylcellulose to each well;

[0078] (4) Cultivation: Place the 96-well cell culture plate in an incubator and culture at 27°C for 43 to 47 hours;

[0079] (5) Cell fixation: Slowly add 150 μl of 80% acetone to each well and fix at room temperature for 30 minutes;

[0080] (6) Washing: discard acetone, pat gently on a paper towel, and wash three times with PBST, 200 μl per well;

[0081] (7) Blocking: Add 50 μl of 30-fold diluted goat serum to each well for blocking and incubate at room temperature with gentle shaking for 5 minutes;

[0082] (8) Add primary antibody: discard the blocking solution, tap 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;

[0083] (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;

[0084] (10) Add secondary antibody: Add 50 μl of HRP-labeled goat anti-mouse diluted antibody working solution to each well and incubate at 37°C for 25 min. HRP-labeled goat anti-mouse antibody was diluted 250 times;

[0085] (11) Washing: discard the secondary antibody, pat gently on a paper towel, and wash three times with PBST, 200 μl per well, 5 minutes each time;

[0086] (12) Color development: Add 50 μl of peroxidase substrate to each well and react at room temperature in the dark for 1-3 hours;

[0087] (13) Microscopic observation: Under optical microscope observation, if no blue spots appear in the normal cell control wells, the test is successful; the number of blue spots in the sample wells to be tested is counted;

[0088] (14) Calculation of virus content: Virus content (IFU / ml) = average number of blue plaques × reciprocal of dilution × 40.

[0089] Fig. 9 This is the status of the cell control and sample wells after staining. It can be seen that there are no non-specific blue spots in the cell control wells, and the blue spots in the virus-infected wells are obvious and the morphology of the blue spots is also very good. This result shows that the screened 7B5-2 monoclonal antibody can be used in the application scenario of determining the baculovirus titer by immunostaining.

[0090] At the same time, the present invention also uses a commercially available anti-gp64 protein polyclonal antibody and a gp64 monoclonal antibody to perform immunostaining according to the above experimental steps to determine the staining of the baculovirus titer. The results are as follows Fig.10 The results show that the blue spots of the gp64 polyclonal antibody-infected wells are identifiable but irregular, and the cell control has more single or small clusters of blue cells; the blue spots of the gp64 monoclonal antibody are poorly morphologically distinguishable, and the cell control is blue overall. It can be seen that the 7B5-2 monoclonal antibody provided by the present invention is significantly superior to the gp64 polyclonal antibody and monoclonal antibody products on the market in the application of immunostaining method to determine the baculovirus titer.

[0091] In addition, according to the above experimental steps, when the 7B5-2 monoclonal antibody was stained, the incubation amount of the antibody was small (only 0.069 μg / well), and the sensitivity was high. 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 determining the baculovirus titer by immunostaining, and the CV of the virus content in three determinations was 12.84% (less than 30%), as shown in Table 2.

[0092] Table 2 Results of three assays of the same batch of baculovirus

[0093]

[0094] The above description of the disclosed embodiments is intended to enable those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 present invention. Therefore, the above drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

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

2.

2. The monoclonal antibody that specifically recognizes baculovirus gp64 protein according to claim 1, characterized in that: It is secreted by the hybridoma cell line 7B5-2, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.46131 and the deposit date November 25, 2024.

3. Use of the monoclonal antibody that specifically recognizes baculovirus gp64 protein according to claim 1 in the preparation of a product for detecting baculovirus.

4. Use of the monoclonal antibody that specifically recognizes baculovirus gp64 protein according to claim 1 in the preparation of a product for determining baculovirus titer by direct immunofluorescence method.

5. A hybridoma cell line 7B5-2 that secretes a monoclonal antibody that specifically recognizes baculovirus gp64 protein, which is deposited with the General Microbiology Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.46131 and a deposit date of November 25, 2024.

6. A kit for determining the titer of baculovirus, characterized in that: The monoclonal antibody comprising the monoclonal antibody specifically recognizing the baculovirus gp64 protein according to claim 1.

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

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