Preparation of cell line for monoclonal antibody against LF protein of Bacillus anthracis, screening of monoclonal antibody, antibody detection kit and application thereof

Abstract

The present invention discloses a preparation of a monoclonal antibody cell line for the LF protein of Bacillus anthracis, monoclonal antibody screening, an antibody detection kit and its application. The nucleic acid sequence of the LF protein is shown in SEQ ID No.1, and the amino acid sequence is shown in SEQ ID No.2. The LF protein monoclonal cell line LF-mAb was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No.45833. The present invention adopts a Bacillus subtilis expression system to prepare a soluble rLF protein with biological activity, obtains a specific monoclonal antibody LF-mAb by immunizing mice and cloning screening, and performs HRP labeling. The rLF protein is used as a coating antigen, and the HRP-labeled specific monoclonal antibody is used as a detection antibody, and an anthrax blocking ELISA antibody detection method is established.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to the preparation of a monoclonal antibody cell line for LF protein of Bacillus anthracis, monoclonal antibody screening, an antibody detection kit and applications thereof. Background Art

[0002] Anthrax is a zoonotic infectious disease caused by infection with Bacillus anthracis. It mainly infects herbivorous animals. Anthrax is still prevalent around the world, which not only affects the development of animal husbandry and the international trade of animal skins, fur, bone meal, etc., but also poses a threat to human health. Humans are mainly infected by contact with livestock fur and meat, ingestion of contaminated food, or by inhalation of dust and aerosols contaminated with anthrax spores.

[0003] Vaccination is an effective means of preventing anthrax. The current veterinary anthrax vaccines mainly include high-temperature attenuated strains and Sterne attenuated strains. Due to the strong pathogenicity of anthrax bacteria, the traditional method of attacking the virus to evaluate the vaccine requires the use of virulent strains, which poses a great biosafety risk and needs to be carried out in a high-level biosafety animal laboratory, which is extremely costly and difficult to carry out. A safe and risk-free immune evaluation method needs to be established. In addition, there is a lack of immune monitoring methods for use in the field, which is not conducive to evaluating the immune level of animal populations.

[0004] The main pathogenic factors of anthrax bacteria include toxins and capsules, whose coding genes are located on two large plasmids of bacteria, pOX1 and pOX2. The toxins cause damage or even death to the host, and the capsule can prevent the bacteria from being phagocytosed by the host's immune cells.

[0005] Anthrax toxin is a typical AB type toxin, which is composed of three factors: Protective Antigen (PA), Leathal Factor (LF) and Edema Factor (EF). They are encoded by the pag, cya and lef genes on the pXO1 plasmid, respectively, and their expression is regulated by the transcriptional activation gene atxA product between cya and pag. The three components are inactive when they exist alone. Only when LF and EF combine with PA to form Leathal Toxin (LT) and Edema Toxin (EF), respectively, and are transported to the cytoplasm through the PA heptamer channel, can the toxin effect be exerted. PA is the binding subunit B of LF / EF, which is responsible for binding to the target cell receptor, and the effector subunit A of LF / EF. LT toxin is the main toxin that causes death in animals. Antibodies against PA and LF can be produced after vaccination. By detecting antibodies against PA or LF, the immunity of anthrax vaccine can be monitored and evaluated.

[0006] International laboratories have successfully expressed LF, but the expression level is very low, only 4%, the purification process is relatively complicated, and there are no reports on the establishment of blocking ELISA methods and reagents for immune evaluation. Therefore, there is a need for a monoclonal antibody cell line preparation, monoclonal antibody screening, antibody detection kit and application of LF protein of Bacillus anthracis. Summary of the invention

[0007] The purpose of the present invention is to provide a cell strain preparation of monoclonal antibody against LF protein of Bacillus anthracis, monoclonal antibody screening, antibody detection kit and application thereof.

[0008] The present invention is achieved through the following technical solutions:

[0009] On the one hand, the LF protein monoclonal antibody cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No.45833.

[0010] Furthermore, LF protein was expressed by Bacillus subtilis, the nucleic acid sequence of the LF protein was shown in SEQ ID No.1, and the amino acid sequence was shown in SEQ ID No.2, a monoclonal antibody cell line was prepared and screened, and the monoclonal antibody was labeled with HRP, and the expressed LF protein was used as the coating antigen by optimizing the conditions.

[0011] Furthermore, the optimized conditions are antigen coating at a protein concentration of 0.25 μg / mL, 100 μL / well, and coating at 4° C. for 12 hours.

[0012] Furthermore, 3% BSA was selected as the blocking solution, and 200 μL / well was blocked at 37°C for 3 hours; positive serum and negative serum were diluted at 1:20 and incubated at 37°C for 1 hour; PBST was washed 3 times, and the enzyme-labeled monoclonal antibody was diluted at 1:3000 and incubated at 37°C for 1 hour; room temperature was used for 10 minutes; after color development, 50 μL / well stop solution was added.

[0013] In another aspect, a kit is provided for detecting anthrax blocking ELISA antibodies, the kit comprising the antibody and a detectable label linked to the antibody.

[0014] Furthermore, the detectable label is an HRP label.

[0015] On the other hand, the LF protein monoclonal antibody cell line or the kit is used in detecting anthrax blocking ELISA antibodies.

[0016] Compared with the prior art, the present invention adopts the above technical solution, and the biggest feature is:

[0017] Compared with the prior art, the present invention has the following effects:

[0018] The invention prepares a soluble rLF protein with biological activity through a Bacillus subtilis expression system, obtains a specific monoclonal antibody cell strain through immunization of mice and clone screening, and HRP labels the monoclonal antibody. The rLF protein is used as a coating antigen, and the HRP-labeled specific monoclonal antibody is used as a detection antibody. An anthrax blocking ELISA antibody detection method is established through condition optimization. The assembled anthrax blocking ELISA antibody detection kit is verified to have good specificity and sensitivity, and a shelf life of at least 9 months. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the pHT43-rLF plasmid map of the present invention;

[0020] Figure 2 This is the SDS-PAGE image of the induction expression of pHT43-rLF of the present invention;

[0021] Where: MW: protein molecular weight standard, 1: empty control, 2: culture supernatant

[0022] Figure 3 This is a western blot identification diagram of pHT43-rLF expression in the present invention;

[0023] Where: MW: protein molecular weight standard, 1: empty control, 2, 3: culture supernatant

[0024] Figure 4 It is the rLF protein purification diagram of the present invention;

[0025] Figure 5 This is a schematic diagram of rLF activity verification of the present invention;

[0026] Figure 6 Schematic diagram of mouse immune serum used in cell fusion experiments for three mice of the present invention;

[0027] Figure 7 This is a schematic diagram of the blocking of the seven monoclonal antibodies of the present invention by anthrax vaccine immune serum;

[0028] Figure 8 A schematic diagram for determining the sensitivity value of the anthrax blocking ELISA antibody detection method of the present invention;

[0029] Fig. 9 A schematic diagram of determining a blocking value of anthrax blocking ELISA antibody detection method according to the present invention; DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is further described below in conjunction with embodiments and comparative examples, but they should not be construed as limiting the present invention:

[0031] Example 1 LF expression and purification

[0032] 1.1 Vector construction and preliminary expression identification

[0033] like Figure 1 As shown, the SPamyQ signal peptide sequence was fused with the LF coding gene and 6His tag by overlap PCR technology to obtain the SPamyQ-LF-6His expression sequence (named slfh). Slfh was cloned into the pHT43 plasmid and named pHT43-rLF.

[0034] Take an appropriate amount of the constructed pHT43-rLF plasmid and add it to the prepared WB800N competent medium, then add 70% PEG600 solution, incubate in an ice bath for 5 minutes, add it to a pre-cooled electroporation cup (1mm), and electroporate once. The electroporation instrument setting: 1.8kV (duration between 4.5ms-5ms);

[0035] Immediately after the electroporation, add 1 ml of recovery medium RM and place at 37°C. After 3 hours of recovery, apply LB plates (containing 5 ug / mL chloramphenicol) and culture the transformed cells overnight at 37°C. The next day, single clones were picked from the above resistance plates for identification.

[0036] Pick a single clone from the plate and add it to 3ml of the corresponding resistance LB medium, and culture it at 37℃ / 200rpm for 3-4h; add 1.5ul IPTG (final concentration 1mM), and induce culture at 37℃ / 200rpm for 4h; collect the inducible bacterial liquid into a 1.5ml EP tube, centrifuge it at 10000rpm for 1min, and keep the supernatant and discard the precipitate. The supernatant sample was tested by SDS-PAGE and identified by western blot.

[0037] 1.2 Expression amplification and protein purification

[0038] like Figure 2 and 3 The expression conditions shown were scaled up and cultured accordingly. After cultivation, the culture was centrifuged at 12,000 rpm for 10 mins, the supernatant was separated, and concentrated 10 times using an ultrafiltration membrane.

[0039] Add a certain amount of treated Nie resin to the concentrated supernatant, incubate in a shaker at 4°C for 30 mins, collect the resin, and purify according to the classic purification method. The purified protein was dialyzed into PBS (pH 7.5), subjected to SDS-PAGE, and the protein concentration was detected. The protein was stored at -80°C for future use. The rLF protein was purified as follows Figure 4 shown.

[0040] 1.3 rLF activity verification

[0041] RAW264.7 cells were plated in 96-well plates at a density of 1*10^5 using DMEM containing 10% heat-inactivated serum, and cell activity was verified after 24 hours.

[0042] Trypsin-digested rPA (prepared in this laboratory, labeled as rPA(T)) and rLF proteins were sterilized by filtration through a 0.22 μm filter membrane. Both rPA(T) and rLF were diluted to 5 ug / ml using DMEM, and then mixed (rPA(T):rLF=1:3). The culture medium of the 96-well cell culture plate was aspirated, and 100 ul / well of the mixed solution was added. Simultaneously, rPA(T) control, rLF, and cell fluid control were made, and incubated at 37°C for 4 hours.

[0043] The incubation solution was aspirated, 100ul DMEM was added to each well, followed by 10ul CCK8 (Solebol) reagent, and the OD450 absorbance was read after incubation at 37°C for 2h. The absorbance was significantly lower than that of the control group (p<0.05), indicating that the cell activity was affected. This proves that the prepared rLF has biological activity. The activity of rLF was verified as follows Figure 5 shown.

[0044] Example 2. Preparation, purification and identification of rLF monoclonal antibody

[0045] 2.1 Immunization of mice

[0046] The purified rLF was emulsified with complete Freund's adjuvant and 60 ug protein / mouse was used for subcutaneous primary immunization of 4 SPF-grade BALB / c female mice, numbered 1, 2, 3, and 4.

[0047] Subcutaneous booster immunization was performed 2, 4, and 6 weeks after the initial immunization, with each immunization dose of 30ug protein / mouse. Blood was collected from the eye sockets 10 days after the third immunization to measure the serum titer. If the titer reached the standard, 50ug of the immunogen was used to intraperitoneally impact the mice 14 days after the third immunization.

[0048] like Figure 6 As shown, immune titer detection: use "rLF", 2ug / ml, 4℃ overnight coating; 5% skim milk, 37℃ blocking for 2h; serum is diluted 2 times from 200 times, blank control is 1*PBS, negative control is negative serum 200 times diluted. Results: The impact #3 mice were selected for cell fusion experiment.

[0049] 2.2 Cell fusion and monoclonal antibody cell line cloning screening

[0050] The spleen cells of mice immunized with rLF were fused with myeloma cells SP2 / 0, and subcloned three times. The indirect ELISA method was used for screening, and 7 positive hybridoma cell lines were obtained. The monoclonal antibody subclasses were identified, and the results are shown in the following table.

[0051]

[0052] 2.3 rLF monoclonal antibody specificity verification

[0053] Use "rLF", 0.2ug / ml, overnight coating at 4℃; 5% skim milk, block at 37℃ for 2h; dilute the anthrax vaccine immune serum twice with 5% skim milk, add 100ul to each well, add 100ul of 5% skim milk to the well without serum, and incubate at 37℃ for 1h. Wash 3 times with PBST, add HRP-labeled goat anti-mouse IgG (10000× dilution) diluted with 5% skim milk, and incubate at 37℃ for 1h. Wash 3 times with PBST, and then add 100ul TMB to each well for color development.

[0054] like Figure 7 As shown in the results, the reaction of the 7 monoclonal antibodies decreased significantly after adding anthrax vaccine immune serum to block, indicating that all 7 monoclonal antibodies are anthrax-specific monoclonal antibodies.

[0055] Example 3 Establishment of Anthrax Blocking ELISA Antibody Detection Method

[0056] 3.1 Monoclonal antibody preparation and labeling

[0057] Cell line No. 39 with the highest blocking rate was selected (biomaterial (strain): LF-mAb; classification and naming: mouse myeloma cell line, LF protein monoclonal antibody cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No. 45833, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), the cells were expanded and cultured, and ascites was prepared. The collected ascites was purified and labeled with HRP enzyme.

[0058] 3.2 Optimization of the optimal reaction conditions for anthrax blocking ELISA antibody detection

[0059] According to the square array titration method, antigen coating was performed at 100 μL / well with 0.5 μg / mL, 0.25 μg / mL, 0.12 μg / mL, and 0.06 μg / mL protein concentrations, and the coating was performed at 4°C for 12 h; PBST was washed 3 times, and the blocking solution was 3% BSA, 5% skim milk, and 3% fish gelatin, and the blocking solution was 200 μL / well at 37°C for 3 h; PBST was washed once, and the positive serum and negative serum were diluted at a ratio of 1:5 and 1:10. , 1:20, 1:40, add 100μL to each well, and act at 37℃ for 1h; wash 3 times with PBST, dilute the enzyme-labeled monoclonal antibody at 1:2000, 1:2500, 1:3000, 1:3500, add 100μL to each well, and act at 37℃ for 1h; wash 3 times with PBST, pat dry, add 100μLTMB to each well, and act at room temperature for 10min; after color development, add 50μL / well stop solution. OD450 value is measured by microplate reader, blocking rate = (negative serum OD450nm value - tested serum OD450nm value) / negative serum OD450nm value × 100%.

[0060] After optimization, the reaction conditions were determined as follows: antigen coating with 0.25 μg / mL protein concentration, 100 μL / well, 4℃ coating for 12h; PBST washing once, 3% BSA blocking solution, 200 μL / well, blocking at 37℃ for 3h; PBST washing 3 times, positive serum and negative serum were diluted at 1:20, 100 μL was added to each well, and 37℃ was used for 1h; PBST washing 3 times, enzyme-labeled monoclonal antibody was diluted at 1:3000, 100 μL was added to each well, and 37℃ was used for 1h; PBST washing 3 times, patting dry, 100 μL TMB was added to each well, and 10min was used at room temperature; after color development, 50 μL / well stop solution was added. The OD450 value was determined by microplate reader, and the blocking rate = (negative serum OD450nm value - tested serum OD450nm value) / negative serum OD450nm value × 100%.

[0061] 3.3 Determination of critical value of anthrax blocking ELISA antibody detection method

[0062] like Figure 8 and 9 As shown, 25 anthrax-negative horse sera (non-immunized) and 30 anthrax vaccine-immunized horse sera (10 immunized for 1 month and 20 immunized for 2 months) were tested by the established blocking ELISA method. The critical value was calculated by the ROC method.

[0063] According to the 95% confidence level analysis, a blocking rate of ≤20% was defined as negative, 20%-30% as suspicious, and ≥30% as positive.

[0064] 3.4 Preparation and shelf life test of anthrax blocking ELISA antibody detection kit

[0065] Antigen coating was performed at 0.25 μg / mL protein concentration, 100 μL / well, at 4°C for 12 h; PBST was used for washing 3 times, 3% BSA + 10% sucrose was selected as the blocking solution, 200 μL / well was used for blocking at 37°C for 3 h, and the samples were blotted dry and then air-dried, and then placed in a sealed bag, and vacuum-sealed after adding a desiccant;

[0066] Add 1‰ proclin to the positive control serum and negative control serum and dispense into 1 ml / tube;

[0067] Add 1‰ proclin to 10× PBST and dispense into 50 ml bottles;

[0068] TMB colorimetric solution, 22 ml / bottle;

[0069] Dispense the stop solution (2 m HCl) into 15 ml / bottle.

[0070] Take 2 96-well antigen-coated plates, 1 tube of positive control serum, 1 tube of negative control serum, 1 bottle of 10×PBST, 1 bottle of TMB colorimetric solution, and 1 bottle of stop solution, assemble into a test kit, store in a 2-8℃ cold storage, and take out a test kit every 3 months for verification. During verification, use the frozen positive immune serum as the standard sample, and set 3 replicates.

[0071] The process is as follows: take out the ELISA plate, wash once with PBST, dilute the positive standard serum, positive control serum and negative control serum at 1:20, add 100 μL to each well, and act at 37°C for 1 hour; wash 3 times with PBST, dilute the enzyme-labeled monoclonal antibody at 1:3000, add 100 μL to each well, and act at 37°C for 1 hour; wash 3 times with PBST, pat dry, add 100 μL TMB to each well, and act at room temperature for 10 minutes; after color development, add 50 μL / well stop solution. OD450 value is measured by microplate reader, blocking rate = (negative serum OD450nm value - test serum OD450nm value) / negative serum OD450nm value × 100%.

[0072] As a result, after the kit was stored for 9 months, the OD value did not decrease significantly, indicating that the kit was stable.

[0073] 3.5 Anthrax Blocking ELISA Antibody Detection Kit Specificity Test

[0074] According to the blocking ELISA method with optimized conditions, the positive serum for equine Salmonellosis, equine glanders, bovine brucellosis, bovine viral diarrhea, and bovine infectious bovine rhinotracheitis were tested, and the test results were all negative.

[0075] 3.6 Anthrax Blocking ELISA Antibody Detection Kit Sensitivity Test

[0076] Three positive sera from horses immunized with anthrax vaccine were serially diluted 2-fold and tested using the established blocking ELISA method to evaluate the sensitivity of the method.

[0077] The results showed that the positive serum of the vaccine-immunized horse could still be detected after dilution 2^6, and was negative after dilution 2^7.

[0078] 3.7 Repeatability test of anthrax blocking ELISA antibody detection kit

[0079] The same batch of ELISA plates coated with the same batch were used to detect the positive sera of vaccine-immunized horses, which was repeated three times, and the intra-batch coefficient of variation was calculated.

[0080] Three different batches of ELISA plates were coated and the positive serum of vaccine-immunized horses was tested. The average value was taken after three repetitions and the coefficient of variation between batches was calculated. Based on the above results, the repeatability of the method was evaluated.

[0081] The results showed that the intra-batch coefficient of variation was less than 1%, and the inter-batch coefficient of variation was less than 3%, both within the acceptable range.

[0082] Intra-assay reproducibility results

[0083]

[0084]

[0085] Batch-to-batch reproducibility results

[0086] Sample No. Repeat 1 Repeat 2 Repeat 3 average value Standard Deviation Coefficient of variation 1 87.026 89.091 87.611 87.909 1.06 1.21％ 2 72.568 73.232 71.495 72.432 0.88 1.21％ 3 42.232 42.532 44.401 43.055 1.18 2.73％

[0087] 3.8 Field Application of Anthrax Blocking ELISA Antibody Detection Kit

[0088] 90 samples of field immune horse serum and bovine serum were collected and tested with anthrax blocking ELISA antibody detection kit. The results showed that the positive rate of horse serum was 88.9% (80 / 90) and the positive rate of bovine serum was 84.4% (76 / 90).

[0089] The present invention successfully prepares soluble rLF protein with biological activity through a Bacillus subtilis expression system, obtains a specific monoclonal antibody cell line through immunization of mice and clone screening, labels LF-mAb with HRP, uses rLF protein as a coating antigen, and uses HRP-labeled specific monoclonal antibody as a detection antibody. Through condition optimization, an anthrax blocking ELISA antibody detection method is established, and an anthrax blocking ELISA antibody detection kit is assembled. It is verified that the kit has good specificity and sensitivity, and a shelf life of at least 9 months.

[0090] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which are all covered by the protection scope of the present invention.

Claims

1. A cell strain of monoclonal antibody against LF protein of Bacillus anthracis, characterized in that: The LF protein monoclonal antibody cell line was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No.45833.

2. A kit for detecting anthrax blocking ELISA antibodies, the kit comprising the LF protein monoclonal antibody cell line according to claim 1 and a detectable marker linked to the LF protein monoclonal antibody.

3. A kit according to claim 2, characterized in that, The detectable label is HRP label.

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