A method for determining baculovirus titer

Abstract

The application discloses a method for determining baculovirus titer, which adopts a mouse IgG2a type monoclonal antibody specifically recognizing envelope protein gp64, and comprises the following steps: step 1, cell plating: cells are plated in a cell culture plate; step 2, baculovirus inoculation: a baculovirus sample to be detected is inoculated into the cell culture plate in step 1; step 3, virus infection; and step 4, flow cytometry detection: infected cells are collected, excessive baculovirus is removed, the cells are washed, fluorescently labeled antibodies are added, and machine detection is carried out. The method of the application is more simple in operation process by using the to-be-detected baculovirus to directly infect insect cells and flow cytometry analysis based on the gp64 antibody, and does not depend on subjective judgment of an experimenter on cytopathic effect, so that the result is more representative.

Description

A method for determining baculovirus titers Technical Field

[0001] This invention belongs to the field of biological detection and analysis technology, and in particular relates to a method for determining the titer of baculoviruses. Background Technology

[0002] The baculovirus expression vector system (BEVS) is a highly efficient eukaryotic expression system for exogenous genes, established based on baculoviruses and their host cells. The most widely used BEVS system utilizes the baculovirus recombinant expression vector derived from the alfalfa silver-striped moth nucleopolyhedrovirus (AcMNPV) through genetic engineering. BEVS is characterized by its safety and stability, high protein expression levels, and post-translational modifications of the expression products, making it a promising candidate for recombinant protein production and the development of AAV vectors for gene therapy.

[0003] When developing BEVS-related methods, both viral amplification and recombinant protein expression require setting the infection ratio of baculovirus to insect cells, i.e., the multiplicity of infection (MOI). Therefore, establishing a rapid and accurate method for determining baculovirus titer is particularly necessary. Currently, commonly used methods for determining baculovirus titer include plaque assay, endpoint dilution assay, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA). The plaque assay is the most classic method. The procedure involves inoculating cells with baculovirus, coating them with agar to prevent viral spread, and culturing for about a week. The viral titer is then quantitatively determined by counting the cytopathic areas formed by viral particles infecting the cells. This method is relatively cumbersome, has a long experimental cycle, and requires a high level of experimental skill from the operator. The endpoint dilution assay uses a 50% tissue culture infectious dose index (TCID). 50 This indicates that directly observing and identifying cellular pathological changes under a microscope, or determining viral infection through fluorescent antibodies, relies heavily on the experience of laboratory personnel, thus human factors can influence the results. Quantitative real-time PCR (qPCR) quantifies the viral genome to infer the baculovirus titer. This method requires the additional step of extracting the viral genome and cannot distinguish between dead viruses and other non-infectious viruses. Summary of the Invention

[0004] Existing methods for determining baculovirus titers suffer from complex and time-consuming procedures (typically requiring 2-7 days), and subjective and experiential factors can influence the results, leading to poor accuracy and repeatability. In view of these shortcomings, the present invention adopts the following specific technical solution:

[0005] In a first aspect of the present invention, a method for determining baculovirus titers is provided, employing a monoclonal antibody of mouse IgG2a type that specifically recognizes the envelope protein gp64, comprising the following steps:

[0006] Step 1, Cell Plating: Plating cells into a cell culture plate;

[0007] Step 2, Baculovirus Inoculation: Inoculate the baculovirus sample to be tested into the cell culture plate from Step 1;

[0008] Step 3, viral infection;

[0009] Step 4, Flow cytometry detection: Collect infected cells, remove excess baculovirus, wash cells, add fluorescently labeled antibodies, and perform flow cytometry detection;

[0010] Furthermore, the sequence of the monoclonal antibody of the mouse IgG2a type that specifically recognizes the envelope protein gp64 is shown in Table 1 below:

[0011] Table 1 Sequence List

[0012]

[0013]

[0014] Furthermore, the method for determining baculovirus titers includes the following steps:

[0015] Step 1, Cell Plating: Plating insect cells in the logarithmic growth phase into a cell culture plate;

[0016] Step 2, Baculovirus Inoculation: The baculovirus sample to be tested is serially diluted and inoculated into the cell culture plate from Step 1.

[0017] Step 3, Viral infection: Place the cell culture plate inoculated with baculovirus in a shaker for incubation;

[0018] Step 4, Flow cytometry detection: Centrifuge to collect the pellet of infected cells, centrifuge to remove excess baculovirus, wash the cells, add fluorescently labeled antibody; after incubation, wash the cells again, transfer them to flow cytometry tubes for detection;

[0019] Step 5, Calculate baculovirus titer: Record the percentage of infected positive cells at each baculovirus dilution, determine the linear range, and calculate the baculovirus titer.

[0020] In some specific embodiments, the insect cells in step 1 are selected from either Sf9 cells or High Five cells, and the density of the insect cells is 4 × 10⁻⁶. 5 ~5×10 5 / ml, each well has a volume of 800μl, and the cell culture plate is a 24-well cell culture plate.

[0021] In some specific implementations, the baculovirus dilution ratio in step 2 is 1:50 to 1:5000. 200 μl of diluted baculovirus sample is added to each well of the cell culture plate, and 2 to 3 replicates are set for each dilution. The control well contains the same volume of fresh culture medium.

[0022] In some specific implementations, in step 3, the cell culture plate inoculated with baculovirus is placed in a shaker at 27°C and cultured at 110 rpm for 6 hours.

[0023] In some specific implementations, step 4 involves collecting infected cells, centrifuging to remove excess baculovirus, washing the cells with PBS, adding a monoclonal antibody of mouse IgG2a type that specifically recognizes the envelope protein gp64, and using a fluorescent label selected from PE, APC, or FITC; incubating at room temperature in the dark for 20 minutes, washing the cells with PBS, and transferring them to flow cytometry tubes for detection.

[0024] In some specific implementations, the formula for calculating the baculovirus titer in step 5 is as follows:

[0025] Baculovirus titer (IU / ml) = Corrected percentage of positive cells × number of cells per well ( / ml) × dilution factor.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] This invention provides a method for determining baculovirus titers by directly infecting insect cells with the baculovirus and using flow cytometry based on a gp64 antibody. It combines the accuracy of cell infection-based assays such as plaque assays and endpoint dilution assays with the rapid and sensitive characteristics of flow cytometry. The procedure is simpler, allowing measurements to be completed within one day. Furthermore, it does not rely on the subjective judgment of cytopathic effects by the experimenter, making the results more representative. By establishing multiple dilutions and selecting appropriate ranges for data averaging, the potential errors from a single experiment can be avoided from significantly affecting the virus titer determination results, thus increasing reliability.

[0028] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0029] Figure 1 shows the results of a flow cytometry experiment that determined the percentage of baculovirus-infected positive cells based on PE fluorescence signals.

[0030] Figure 2 shows the relationship between the dilution factor and the percentage of positive cells after correction. Detailed Implementation

[0031] To make the technical means, inventive features, objectives, and effects of the invention readily understandable, the invention is further illustrated below with reference to specific figures. However, the invention is not limited to the embodiments described below.

[0032] It should be noted that the structures, proportions, sizes, etc., illustrated in the accompanying drawings of this specification are only used to complement the content disclosed in the specification for those skilled in the art to understand and read, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0033] Unless otherwise listed, all reagents, antibodies, and consumables used in the following experimental examples and embodiments are commercially available products.

[0034] (1) The EZNAEndo-Free BAC / PAC DNA Isolation Kit was purchased from Omega.

[0035] (2) Sf9 insect cells and Sf-900 II SFM medium were purchased from Gibco.

[0036] (3) High Five insect cells and Express Five SFM medium were purchased from Gibco.

[0037] (4) Grace's Insect Medium was purchased from Gibco.

[0038] (5) Cellfectin II Reagent was purchased from Invitrogen.

[0039] (6) The LSL Fortessa Cell Analyzer was purchased from BD.

[0040] Experiment 1: Extraction of Bacmid Plasmid

[0041] (1) Transform DH10Bac competent cells with pFastBac recombinant plasmid carrying the target gene MEK1 and culture them on LB blue-white screening plates containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 7 μg / ml tetracycline, 40 μg / ml IPTG and 100 μg / ml X-gal. Incubate at 37℃ in the dark for 48 h until clear blue-white colonies appear on the plates.

[0042] (2) Use a sterilizing pipette tip to pick up a single white colony with a regular circular edge, streak it on a new LB blue-white screening plate, and incubate it in a constant temperature incubator at 37°C in the dark for 24 hours.

[0043] (3) Pick a single white colony from the above secondary screening plate and inoculate it into LB liquid medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin and 7 μg / ml tetracycline. Incubate at 37°C and 250 rpm for 3 h until the bacterial solution becomes turbid. Further verify the singleness of the picked colony by PCR.

[0044] (4) Use the EZNAEndo-Free BAC / PAC DNA Isolation Kit to extract the recombinant baculovirus plasmid (Bacmid DNA) and store it at -20℃ for later use.

[0045] Experimental Example 2: Preparation of P1 generation baculovirus

[0046] (1) Cell culture: Sf9 insect cells and Sf-900 II SFM medium were used for amplification and passage in suspension culture mode;

[0047] (2) Take 1×10 6 ~1.2×10 6 Sf9 cells at a density of / ml were seeded at 2ml per well in a six-well cell culture plate and incubated overnight at 27°C to allow the cells to adhere fully.

[0048] (3) When the cell confluence reaches 85% to 95%, perform transfection, discard the old culture medium, add 2 ml of Grace's Insect Medium to each well, and place in a 27°C incubator for 1 hour.

[0049] (4) Prepare the transfection mix:

[0050] Mix 1:100μl Grace's Insect Medium+1.5μg Bacmid DNA

[0051] Mix 2:100μl Grace's Insect Medium+3μl Cellfectin II Reagent

[0052] Combine the two Mixes mentioned above, mix well, and let stand at room temperature in the dark for 30 minutes.

[0053] (5) Add the transfection Mix dropwise to each well of the six-well plate, place it in a 27°C incubator and let it stand for 6 hours. Discard the supernatant and replace it with 2 ml of Sf-900 II SFM medium. Continue to incubate in a 27°C incubator.

[0054] (6) Obvious changes in cell morphology can be observed 72 hours after transfection. After culturing for 5-7 days, P1 generation baculovirus is harvested, the supernatant is aspirated, centrifuged at 1000 rpm for 5 min, filtered through 0.22 μm and stored at 4℃ in the dark.

[0055] Example

[0056] Viral infection

[0057] (1) Cell culture: High Five insect cells and Express Five SFM medium were used for amplification and passage in suspension culture mode;

[0058] (2) Cell plating: High Five cells in the logarithmic growth phase were plated at a density of 4 × 10⁻⁶ cells / mL. 5 The cells were seeded at a density of 800 μl / ml in a 24-well cell culture plate, with each well containing 800 μl.

[0059] (3) Inoculation with baculovirus: The baculovirus samples to be tested were serially diluted according to Table 2 using Sf-900II SFM medium, which is consistent with the virus packaging process. The samples were then inoculated into the culture plates containing cells. 200 μl of diluted virus sample was added to each well. 2 to 3 replicates were set for each dilution. The control wells contained the same volume of fresh culture medium.

[0060] Table 2. Serial dilution method for baculovirus samples

[0061]

[0062] (4) Viral infection: The cell culture plate inoculated with baculovirus was placed in a shaker at 27°C and cultured at 110 rpm for 6 h;

[0063] (5) Flow cytometry detection: Infected cells were collected into 1.5 ml centrifuge tubes, centrifuged at 1000 rpm for 3 min to remove excess baculovirus, and washed with 1 ml PBS buffer. PE-labeled monoclonal antibody that specifically recognizes the envelope protein gp64 (its heavy and light chain sequences are shown in Table 1) was added, with 0.125 μg of monoclonal antibody used for each sample. The cells were incubated at room temperature in the dark for 20 min. After washing the cells with 1 ml PBS, the cells were resuspended in 200 μl PBS and transferred to flow cytometry tubes. Data were collected using an LSR Tortessa Cell Analyzer.

[0064] Comparative Example

[0065] In the examples, another gp64 antibody was selected for simultaneous control experiments.

[0066] Candidate antibody sample preparation method:

[0067] (1) Antigen preparation (gp64 recombinant protein): A His6 tag was fused to the amino acid sequence corresponding to the extracellular region (Ala21-Thr481) of gp64 protein. After gene synthesis, it was secreted and expressed through an insect cell expression system. The protein was purified by immobilized metal ion affinity chromatography to obtain gp64 recombinant protein with a purity >85% as an antigen for mouse immunization.

[0068] (2) Animal immunization: 8-10 week old female BALB / c mice were immunized three times via intraperitoneal injection at time points d1, d15 and d29. Each time, 120 μg of recombinant gp64 protein and 100 μl of adjuvant were used (the protein was diluted with physiological saline, and the total injection volume was 200 μl). The amount of recombinant protein injected in the first immunization was twice that of all subsequent immunizations. When the mice's serum ELISA turned positive, indicating that the antibody titer was sufficient, the mice were boosted with recombinant protein antigen (without adjuvant, and the volume was increased to 200 μl with physiological saline). Blood was collected for cell fusion 3 days later.

[0069] (3) Fusion and screening: Spleen cells of mice were isolated from blood samples of immunized mice, and a suspension of spleen cells was prepared. The suspension was mixed with myeloma cells F0 at a ratio of 2:1 and fused using an electrofusion instrument. The fused cells were transferred into HAT screening medium, and monoclonal antibodies were obtained by limiting dilution. Candidate antibodies were screened by ELISA and flow cytometry.

[0070] (4) Antibody purification: The candidate monoclonal cells were expanded and cultured. The cell supernatant was purified in one step using Protein A packing material. After concentration and medium exchange, the supernatant was stored in PBS with a concentration of more than 1 mg / ml for subsequent testing.

[0071] The test results for the comparative model are shown in Table 7.

[0072] Calculating baculovirus titers

[0073] (1) Data analysis was performed using FlowJo software. Positive gates were identified by the PE fluorescence signal value of the baculovirus-free control group (Figure 1, corresponding to serial numbers 0 and 1 of the first experiment). The percentage of positive cells infected with baculovirus samples at different dilutions (%gated, Table 3) was obtained.

[0074] Table 3. Percentage of positive cells infected with baculovirus at different dilutions

[0075]

[0076] (2) The linear range of variation (Figure 2) was determined based on the correspondence between the dilution factor and the corrected percentage of positive cells, and was numbered 4 to 8. The specific method was as follows: groups with a corrected percentage of positive cells <1%, i.e., groups numbered 9 to 11, were discarded; starting from group number 8, groups were selected one by one towards smaller numbers until the linear regression determination coefficient could not meet R. 2 The requirement of >0.9 (while discarding all other groups above it and not participating in subsequent calculations) means that the groups with serial numbers 4 to 8 finally meet the condition.

[0077] The baculovirus titer was calculated at each dilution (Table 4), and the average value was considered the final titer value for the baculovirus sample. The formula for baculovirus titer is:

[0078] Baculovirus titer (IU / ml) = Corrected percentage of positive cells × Number of cells per well ( / ml) × Dilution factor

[0079] Table 4 Results of Baculovirus Titer Determination

[0080]

[0081] (3) The same rod-shaped virus sample was retested using the above method, and the data obtained are shown in Table 5, indicating that the method has high reproducibility (Table 6).

[0082] Table 5 Results of repeated determinations of baculovirus titer

[0083]

[0084] Table 6 Comparison of the two results

[0085]

[0086] Table 7 Results of Baculovirus Titer Determination

[0087]

[0088] The above experimental results show that when another gp64 antibody was used as a control in the examples, it was found that the antibody performed worse in terms of sensitivity or specificity than the monoclonal antibody in this patent, resulting in a lower measured baculovirus titer.

[0089] This invention uses gp64 fluorescent antibody to directly identify insect cells infected with a virus and collects the signal using flow cytometry. Compared to a technical solution that constructs a specialized recombinant vector to fuse a specific gene into a target gene and then detects the signal using flow cytometry, this method eliminates the need for a dedicated recombinant vector, thus reducing workload. Furthermore, the aforementioned technical solutions only measure the titer of recombinant baculoviruses carrying specific genes and cannot effectively reflect the titer level of corresponding baculoviruses without specific genes. The technical solution of this invention has better advantages.

[0090] This invention uses gp64 fluorescent antibody to identify virus-infected insect cells. Through flow cytometry, experimental studies were conducted on infection ratio gradient settings, infection time, cell culture methods, data processing and interpretation methods. Targeted experimental design was carried out for flow cytometry application scenarios, and good experimental results were obtained.

[0091] The present invention has the following beneficial effects:

[0092] This invention provides a method for determining baculovirus titers by directly infecting insect cells with the baculovirus and using flow cytometry based on a gp64 antibody. It combines the accuracy of cell infection-based assays such as plaque assays and endpoint dilution assays with the rapid and sensitive characteristics of flow cytometry. The procedure is simpler, allowing measurements to be completed within one day. Furthermore, it does not rely on the subjective judgment of cytopathic effects by the experimenter, making the results more representative. By establishing multiple dilutions and selecting appropriate ranges for data averaging, the potential errors from a single experiment can be avoided from significantly affecting the virus titer determination results, thus increasing reliability.

[0093] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for determining baculovirus titers, characterized in that, The method employs a mouse IgG2a type monoclonal antibody that specifically recognizes the envelope protein gp64, comprising the following steps: Step 1, cell plating: plating cells into a cell culture plate; Step 2, baculovirus inoculation: inoculating the baculovirus sample to be tested into the cell culture plate from Step 1; Step 3, virus infection; Step 4, flow cytometry detection: collecting infected cells, washing cells, adding fluorescently labeled antibody, and performing flow cytometry detection; the mouse IgG2a type monoclonal antibody that specifically recognizes the envelope protein gp64 comprises: heavy chain variable region CDR1, the sequence of which is shown in SEQ ID NO. 3; heavy chain variable region CDR2, the sequence of which is shown in SEQ ID NO. 4; heavy chain variable region CDR3, the sequence of which is shown in SEQ ID NO. 5; light chain variable region CDR1, the sequence of which is shown in SEQ ID NO. 8; light chain variable region CDR2, the sequence of which is shown in SEQ ID NO. 9; and light chain variable region CDR3, the sequence of which is shown in SEQ ID NO.

10.

2. The method for determining baculovirus titer according to claim 1, characterized in that, The monoclonal antibody of the mouse IgG2a type that specifically recognizes the envelope protein gp64 includes: a heavy chain variable region, the sequence of which is shown in SEQ ID NO.2; and a light chain variable region, the sequence of which is shown in SEQ ID NO.

7.

3. The method for determining baculovirus titer according to claim 2, characterized in that, The monoclonal antibody of the mouse IgG2a type that specifically recognizes the envelope protein gp64 comprises: a heavy chain, the sequence of which is shown in SEQ ID NO.1; and a light chain, the sequence of which is shown in SEQ ID NO.

6.

4. The method for determining baculovirus titer according to claim 1, characterized in that, The procedure includes the following steps: Step 1, cell plating: Insect cells in the logarithmic growth phase are plated in a cell culture plate; Step 2, baculovirus inoculation: The baculovirus sample to be tested is serially diluted and inoculated into the cell culture plate from Step 1; Step 3, virus infection: The cell culture plate inoculated with baculovirus is placed in a shaker for culture; Step 4, flow cytometry detection: The infected cells are collected by centrifugation, washed, and fluorescently labeled antibodies are added; after incubation, the cells are washed again and transferred to flow cytometry tubes for detection; Step 5, baculovirus titer calculation: The percentage of infected positive cells at each baculovirus dilution is recorded, the linear range is determined, and the baculovirus titer is calculated.

5. The method for determining baculovirus titer according to claim 4, characterized in that, The insect cells used in step 1 are selected from either Sf9 cells or High Five cells, and the density of the insect cells is 4 × 10⁻⁶. 5 ~5×10 5 / ml, with a volume of 800 μl per well, and the cell culture plate is a 24-well cell culture plate.

6. The method for determining baculovirus titer according to claim 4, characterized in that, In step 2, the baculovirus dilution ratio is 1:50 to 1:5000. 200 μl of diluted baculovirus sample is added to each well of the cell culture plate, and 2 to 3 replicates are set for each dilution. The control well contains the same volume of fresh culture medium.

7. The method for determining baculovirus titer according to claim 4, characterized in that, In step 3, the cell culture plate inoculated with baculovirus is placed in a shaker at 27°C and cultured at 110 rpm for 6 h.

8. The method for determining baculovirus titer according to claim 4, characterized in that, Step 4 involves collecting infected cells, centrifuging to remove excess baculovirus, washing the cells with PBS, adding a monoclonal antibody of mouse IgG2a type that specifically recognizes the envelope protein gp64, and using a fluorescent label selected from PE, APC, or FITC; incubating at room temperature in the dark for 20 minutes, then washing the cells with PBS and transferring them to flow cytometry tubes for detection.

9. The method for determining baculovirus titer according to claim 4, characterized in that, The formula for calculating the baculovirus titer in step 5 is: Baculovirus titer IU / ml = Corrected percentage of positive cells × Number of cells per well / ml × Dilution factor.

Images