[0015] Example 1 Preparation and identification of monoclonal antibodies of the present invention
[0016] 1. Preparation of monoclonal antibody 3A6
[0017] 1) Design, preparation and carrier coupling of Toxoplasma gondii TgVP1 antigen peptide
[0018] According to the TgVP1 sequence of Toxoplasma gondii on GenBank (accession number: EPT25031.1), the TgVP1 protein sequence of Toxoplasma gondii contains 816 amino acids. The TMHMM software was used to analyze the transmembrane domain of the TgVP1 protein, and the IEDB software was used to analyze the immunogenicity, hydrophobicity and surface accessibility of the TgVP1 protein. The results showed that the Toxoplasma gondii TgVP1 protein 292-320aa has 29 amino acid antigenicity, hydrophilicity and surface accessibility. Strong accessibility. After the above analysis, the final screening polypeptide sequence is: 292aa-320aa, SEQ ID NO: 2. The short peptide (TgVP1-P1) and the C-terminal short peptide (TgVP1-P1-KLH) coupled with the keyhole limpet carrier protein KLH (TgVP1-P1-KLH) were synthesized by Shanghai Jier Biochemical Company.
[0019] 2) Immunize mice
[0020] The synthetic peptide TgVP1-P1-KLH was used to immunize female BALB/c mice aged 6-8 weeks.
[0021] The first immunization: After mixing the synthetic peptide TgVP1-P1-KLH with an equal volume of Bentonite adjuvant, inject BALB/c mice intraperitoneally with an amount of 50μg/500μL each;
[0022] Second immunization: After 2 weeks, take the synthetic peptide TgVP1-P1-KLH and mix it with an equal volume of Bentonite adjuvant, and then inject BALB/c mice intraperitoneally with an amount of 50μg/500μL each;
[0023] Third immunization: After another 2 weeks, take the synthetic peptide TgVP1-P1-KLH and equal volume of Bentonite adjuvant and mix them, and then inject BALB/c mice intraperitoneally at the amount of 50μg/500μL each; the third immunization After 10 days, blood was taken from the tail vein of the mouse and coated with synthetic peptide TgVP1-P1. The serum titer was detected by ELISA, and the titer was greater than 1:10 5 Fusion of mouse spleen cells with myeloma cells;
[0024] Bentonite adjuvant uses commercial products.
[0025] 3) Determination of immune serum titer
[0026] The indirect ELISA method was used to determine the titer of immune serum. Take 50μg of synthetic peptide TgVP1-P1 and dissolve it in 10ml of 0.05M pH9.6 carbonate buffer, and coat it in a 96-well polystyrene microplate, 100μl/well, overnight at 4°C. Wash the plate three times with PBS (containing 0.05% (V/V) Tween-20), use 10mM PBS containing 1% BSA blocking solution 100μl/well, block at 37°C for 2h, use PBS (containing 0.05% (V/V) Tween- 20) The plate was washed three times, and blood was collected from the tail vein of the mouse 10 days after the third immunization. The mouse immune serum was subjected to 10 mM PBS containing 1% BSA. -2 ~10 -8 Double dilution, add to 96-well plate, 100μl/well 37℃1h, PBS (containing 0.05% (V/V) Tween-20) wash the plate three times, add 1:10000 times diluted horseradish peroxidase labeled goat anti-small Mouse IgG (Sigma, INC.), 100μl/well at 37℃ for 30min, same as above, after washing the plate, TMB develops color, 100μl/well, at room temperature for 10min, add 50μl/well 2MH 2 SO 2 Stop the reaction, measure the 450nm absorbance value, use the pre-immune mouse serum as a negative control, and use the ratio of the measured value to the control value ≥ 2.1 as positive to judge the titer of the immune serum.
[0027] 4) Preparation of hybridoma
[0028] Take serum titer greater than 1:10 5 Three days before fusion, the synthetic peptide TgVP1-P1 was mixed with an equal volume of PBS, and then BALB/c mice to be fused were injected intraperitoneally with 50μg/500μL each for booster immunization. Take the mouse spleen aseptically, prepare a spleen cell suspension and mix with logarithmic growth phase mouse myeloma cell line SP2/0 at a ratio of 1:1, centrifuge at 1000×g at room temperature for 5 minutes, discard the supernatant, and gently Flick the bottom of the centrifuge tube to loosen the precipitate. Place the centrifuge tube in a 37°C water bath. Add 50% polyethylene glycol (PEG, MW4000, Sigma) that is kept in the 37°C water bath into the centrifuge tube drop by drop. While shaking the centrifuge tube, drip in 1 min. After dripping, let it stand for 2 min. Add 1ml, 2ml, 3ml, 4ml, 5ml and 10ml of serum-free 1640 medium preheated at 37℃ every 1 minute to stop polyethylene glycol. Effect: Centrifuge the cell mixture at 1000×g for 5 min at room temperature, discard the supernatant, add HAT medium (hypoxanthine (H), aminopterin (A) and thymidine (T) (HAT, Sigma)) and gently resuspend Cells, divide the cells into 96-well plates, 200μl per well. After culturing for three days, observe the cell fusion and replace half of the HAT medium for several consecutive days until a clone is formed. Change the HT medium (hypoxanthine (H) and thymidine (T)) (HT, Sigma ))to cultivate.
[0029] 5) Screening of hybridoma cells that secrete anti-TgVP1 monoclonal antibodies
[0030] The cell culture supernatant was screened by indirect ELISA, and the positive clone hybridoma cells with higher titer were selected for subcloning, and the limiting dilution method was used for continuous cloning 2-3 times until the positive rate of 100% cells was reached, and finally a stable secreting antibody was obtained. TgVP1 monoclonal antibody cell line, labeled 3A6. The cloned cells with a positive rate of 100% were expanded and cultured and then frozen in liquid nitrogen.
[0031] 6) Preparation and purification of ascites
[0032] The 3A6 hybridoma cell line was divided into 1×10 6 The amount per mouse is injected into the abdominal cavity of 8-10 weeks old BALB/c female mice pretreated with liquid paraffin, and ascites is drawn when the mice’s abdomen is enlarged for 10-14 days after feeding and observation. The monoclonal antibody was purified by affinity chromatography Protein G Sepharose Fast Flow, and the purity of the monoclonal antibody was determined by SDS-PAGE, and the purity reached more than 90%.
[0033] 2. Characterization of the monoclonal antibody of the present invention
[0034] 1) Determination of antibody concentration: Ascites prepared by hybridoma cell CCTCC NO: C2014231 is purified to obtain TgVP1 monoclonal antibody 3A6, which is determined by Smart Spec plus nucleic acid protein analyzer produced by BIO-RAD, and its concentration is 1.24mg/ ml.
[0035] 2) Antibody subtype identification: Roche's mouse monoclonal antibody subtype identification kit is used to identify the subtype of the hybridoma cell line. The subtype of the 3A6 secreted antibody is IgG1 type, and the light chain is κ chain.
[0036] 3) Identification of the titer of the purified antibody: 50 μg of synthetic peptide TgVP1-P1 was dissolved in 10 ml of pH 9.6 0.05M carbonate coating buffer, added to a 96-well plate, 100 μL per well, overnight at 4°C. Wash the plate three times with PBS (containing 0.05% (V/V) Tween-20), use 10mM PBS containing 1% BSA blocking solution 150μl/well, block at 37℃ for 2h, use PBS (containing 0.05% (V/V) Tween-20) ) Wash the plate three times, add 100μl purified antibody to each well, incubate at 37°C for 1h, wash the plate three times with PBS (containing 0.05% (V/V) Tween-20), add horseradish peroxidase labeled goat anti-mouse IgG polyclonal The antibody is a secondary antibody, incubate at 37°C for 30min, wash the plate three times with PBS (containing 0.05%(V/V)Tween-20), add 100μl to each well, develop color with TMB, incubate at 37°C for 15min, add 2MH 2 SO 4 The solution terminates the reaction, and the microplate reader detects the absorbance at 450nm.
[0037] 4) Western blot identification of antibodies: Toxoplasma gondii tachyzoites and OFTu cells were collected and lysed into Toxoplasma gondii lysate protein and OFTu lysate protein, lysed with 2×SDS lysis buffer and loaded, and subjected to 10% SDS -After PAGE, use the Bio-Rad electro-transfer device to transfer the protein to the PVDF membrane, block with 5% skimmed milk powder for 1 hour, and wash the membrane with a pH 7.4 Tris-HCl buffer (containing 0.1% (V/V) Tween-20) 3 The anti-TgVP1 monoclonal antibody 3A6 prepared by purified hybridoma cell CCTCCNO: C2014231 was added at 1:1000 times, 5 minutes each time, and incubated overnight at 4°C, pH 7.4 Tris-HCl buffer (containing 0.1% (V/V) )Tween-20) Wash the membrane 3 times, 5min each time, add 1:10000 diluted goat anti-mouse IgG polyclonal antibody (Sigma) as the secondary antibody, incubate for 2h at room temperature, wash the membrane 3 times with TBST, absorb the excess with filter paper Spread the solution flat on clean plastic wrap, add 1.4ml Pierce-Thermo Scientific ECL series Western chemiluminescence substrate reaction solution (A:B=1:1) to completely soak the film in the reaction solution, take it out quickly, and use Filter paper to absorb excess liquid, spread it on another piece of cling paper, wrap the film with cling paper, put it into an X-ray photography cassette, and develop it in a darkroom. The anti-TgVP1 monoclonal antibody 3A6 prepared by the hybridoma cell CCTCC NO: C2014231 showed a single specific band for the toxoplasma lysate protein. The results are as follows figure 1 Shown.
[0038] 5) Immunofluorescence identification of antibodies: prepare Toxoplasma gondii smears, fix with 4% paraformaldehyde for 15 minutes, add 0.25% TritonX100 dropwise, and incubate for 10 minutes at room temperature to make the cell membrane permeable for the antibody to enter. Wash with PBS three times and block with PBST containing 1% BSA for 2 hours at room temperature. Washed with PBS for 3 times, and then incubated with the anti-TgVP1 monoclonal antibody 3A6 (PBST 1:500 dilution) of the present invention for 1.5 hours at room temperature. Washed with PBS 3 times, and incubated with Alexa Fluor 546 (red fluorescence) labeled goat anti-mouse IgG (PBST 1:1000 dilution) for 1 hour at room temperature. DAPI staining (blue) 10min after washing with PBS. After washing the film with PBS, observe it under a fluorescence microscope. At the same time, normal mouse serum was used as a negative control.
[0039] Results: Red fluorescence was observed in the Toxoplasma gondii cytoplasm stained with the anti-TgVP1 monoclonal antibody 3A6 prepared by the hybridoma cell CCTCC NO: C2014231 of the present invention, which proved that the anti-TgVP1 antibody of the present invention can recognize the natural TgVP1 protein.
[0040]
[0041]
