Preparation method of echinococcus multilocularis recombinant P29 protein monoclonal antibody

Abstract

The embodiment of the invention provides a preparation method of an echinococcus multilocularis recombinant P29 protein monoclonal antibody, and relates to the technical field of preparation of P29 protein monoclonal antibodies, and the preparation method comprises the following steps: feeding experimental animals; constructing and purifying the recombinant protein P29; preparation and application of the monoclonal antibody; preparing hybridoma cells; according to the application of the monoclonal antibody, rEm.P29 is used for immunizing BALB / c mice, hybridoma cells are prepared, the recombinant protein P29 monoclonal antibody is screened out, the recombinant protein P29 monoclonal antibody can inhibit the growth of the echinococcus multilocularis in vitro, and the prepared monoclonal antibody has the advantages of being high in sensitivity, affinity and specificity and the like besides inhibiting the growth of the echinococcus multilocularis.

Description

Technical Field

[0001] The present application relates to the technical field of preparation of monoclonal antibodies against P29 protein, and in particular to a method for preparing monoclonal antibodies against recombinant P29 protein of Echinococcus multilocularis. Background Art

[0002] The morphology and life history of Echinococcus multilocularis are similar to those of Echinococcus granulosus, but the adult worm mainly parasitizes foxes, the intermediate host is rodents or insectivores, and the larval stage is Echinococcus multilocularis (also known as alveolar cysts). It causes severe alveolar echinococcosis in humans, also known as alveolar echinococcosis, or multilocular echinococcosis. Human alveolar echinococcosis caused by Echinococcus multilocularis infection is a serious zoonotic parasitic disease, and its pathogen is mainly transmitted to humans through ingestion of food or water containing infectious eggs. Monoclonal antibodies, as a new type of therapeutic tool, have shown great potential in the field of precision medicine. Especially for the diagnosis and treatment of certain diseases, monoclonal antibodies have become a hot topic of research due to their high specificity, high affinity and repeatability.

[0003] Currently, there are limited treatment options for alveolar echinococcosis, which usually require surgical removal of the diseased tissue combined with drug treatment such as albendazole. However, the therapeutic effect is not ideal and there is a risk of recurrence. It is impossible to develop antibodies against Echinococcus multilocularis to inhibit the growth of Echinococcus multilocularis larvae, which brings inconvenience to the treatment of the disease. Summary of the invention

[0004] The present application aims to solve at least one of the technical problems in the prior art that antibodies developed from Echinococcus multilocularis can not be used to inhibit the growth of Echinococcus multilocularis larvae. To this end, the present application proposes a method for preparing a monoclonal antibody against a recombinant P29 protein of Echinococcus multilocularis.

[0005] According to an embodiment of the present application, a method for preparing a monoclonal antibody against a recombinant P29 protein of Echinococcus multilocularis comprises the following steps:

[0006] S1. Animal husbandry: The experimental animals were 10 6-8 week old female Balb / c mice;

[0007] S2. Construction and purification of recombinant protein P29, wherein the steps include:

[0008] 1) Cultivation of rEg.P29 recombinant strain and induced expression of P29 protein;

[0009] 2) Purification of rEgP29;

[0010] 3) Protein dialysis;

[0011] 4) Determination of protein concentration by BCA method;

[0012] S3. Preparation and application of monoclonal antibodies, wherein the steps include:

[0013] 1) Immunization regimen;

[0014] 2) Immunization method;

[0015] 3) Determination of serum antibody titer of immunized mice;

[0016] S4, hybridoma cell preparation, wherein the steps include:

[0017] 1) Myeloma cell preparation;

[0018] 2) Preparation of spleen cells;

[0019] 3) Cell fusion;

[0020] 4) Screening of fusion cells;

[0021] 5) Cell subcloning and subclone screening;

[0022] S5. Application of monoclonal antibodies, wherein the steps include:

[0023] 1) Monoclonal antibody cell line subtype determination;

[0024] 2) Determination of monoclonal antibody titer;

[0025] 3) Purification of monoclonal antibodies;

[0026] 4) Evaluation of monoclonal antibodies on the infection model prepared by intraperitoneal injection of alveolar protoscolex in mice;

[0027] 5) Evaluation of 1G6F7 monoclonal antibody on the infection model prepared by portal vein injection of alveolar protoscolex in mice.

[0028] Preferably, in S2, the specific steps of culturing the rEg.P29 recombinant strain and inducing expression of the P29 protein include:

[0029] S1, single colony culture;

[0030] S2, induced expression;

[0031] S3, collect bacteria and prepare samples;

[0032] S4. SDS-PAGE detection of target protein. The specific steps include:

[0033] 1) Preparation of lower separation gel;

[0034] 2) Preparation of upper concentrated gel;

[0035] 3) Add sample;

[0036] 4) Electrophoresis;

[0037] 5) Coomassie Brilliant Blue staining;

[0038] 6) Decolorization;

[0039] 7) Take photos.

[0040] Preferably, in said S2, the specific steps of purifying rEgP29 include:

[0041] S1, big shake;

[0042] S2, collecting bacteria;

[0043] S3, schizobacterium;

[0044] S4. Purification.

[0045] Preferably, in S2, the specific steps of protein dialysis include:

[0046] S1, dialysis bag activation;

[0047] S2, dialysis;

[0048] S3. Collect protein.

[0049] Preferably, in S2, the specific steps of determining the protein concentration by the BCA method include:

[0050] S1. Prepare BCA working solution;

[0051] S2, prepare standard porins;

[0052] S3, sample dilution;

[0053] Add 200 μL / well of BCA working solution to S4, standard wells and sample wells, mix well and incubate at 37°C for about 15 min.

[0054] S5. OD value determination;

[0055] S6. Draw a standard curve to calculate the protein concentration, and store the protein in aliquots at -80°C for later use.

[0056] Preferably, in said S3, the specific steps of determining the serum antibody titer of the immunized mice include:

[0057] S1, collecting serum from the tail vein of mice;

[0058] S2. The ELISA method was used to detect the P29-specific antibody titer in the sera of 5 mice.

[0059] Preferably, in S5, the hybridoma cells are subcloned by limiting dilution method, and after detection to 100% positive, single clone wells are picked out for expansion culture and determination of strains for subclone screening.

[0060] Preferably, in S5, both fusion cell screening and subclone screening are screened and detected by an indirect ELISA method.

[0061] Preferably, in S5, the monoclonal antibody is purified using Protein G agarose gel medium and detected, and the infection model is established by injecting alveolar protoscolex into the peritoneal cavity or portal vein of mice.

[0062] Preferably, in said S5, the evaluation steps of the infection model prepared by intraperitoneal injection of monoclonal antibody to mice and the evaluation steps of the infection model prepared by portal vein injection of monoclonal antibody 1G6F7 to mice are:

[0063] S1. Isolation and activity detection of alveolar protoscolex, the method is the same as the determination of serum antibody titer of immunized mice;

[0064] S2. Preparation of mouse intraperitoneal injection of alveolar protoscolex infection model;

[0065] S3. Grouping and intervention.

[0066] The beneficial effects of the present application are as follows: the present invention utilizes rEm.P29 to immunize BALB / c mice, prepares hybridoma cells, and screens out recombinant protein P29 monoclonal antibodies. The recombinant P29 monoclonal antibodies can inhibit the growth of multilocular echinococcosis in vitro. In addition to inhibiting the growth of multilocular echinococcosis, the prepared monoclonal antibodies also have the characteristics of high sensitivity, high affinity, and strong specificity.

[0067] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0069] Figure 1 This is a detection diagram of protein induced expression detected by SDS-PAGE according to the embodiment of the present application.

[0070] Figure 2 1 is a flow chart of the preparation of monoclonal antibodies according to an embodiment of the present application.

[0071] Figure 3 This is a graph showing the results of detecting anti-Em.P29 specific antibodies in the sera of immunized mice according to an embodiment of the present application.

[0072] Figure 4 This is a diagram of ELISA detection of anti-Em.P29 specific antibodies in culture supernatant according to the example of the present application.

[0073] Figure 5 This is a diagram of anti-Em.P29 specific antibodies in the culture supernatant of positive cell lines screened by subcloning according to the embodiment of the present application.

[0074] Figure 6 This is a graph showing the antibody titer detection of 4 monoclonal antibodies according to the examples of the present application.

[0075] Figure 7 This is a graph evaluating the therapeutic effect of the 1G6F7 monoclonal antibody according to the examples of the present application on the mouse model of intraperitoneal infection of Echinococcus multilocularis.

[0076] Figure 8 This is a graph evaluating the therapeutic effect of the 1G6F7 monoclonal antibody according to the examples of the present application on the mouse model of Echinococcus multilocularis portal vein infection. DETAILED DESCRIPTION

[0077] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application.

[0078] In order to make the purpose, technical solutions and advantages of the implementation methods of this application clearer, the technical solutions in the implementation methods of this application will be clearly and completely described below in conjunction with the drawings in the implementation methods of this application. Obviously, the described implementation methods are part of the implementation methods of this application, not all of the implementation methods. Based on the implementation methods in this application, all other implementation methods obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0079] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0080] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0081] In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicating orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0082] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the following steps. In the description of this application, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0083] In this application, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to specific circumstances.

[0084] In the present application, unless otherwise clearly specified and limited, the first feature being "above" or "below" the second feature may include the following steps: the first and second features are in direct contact, or may include the following steps: the first and second features are not in direct contact but are in contact through another feature between them. Moreover, the first feature being "above", "above" and "above" the second feature includes the following steps: the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. The first feature being "below", "below" and "below" the second feature includes the following steps: the first feature is directly below and obliquely below the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0085] The following is a description of a method for preparing a monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to an embodiment of the present application with reference to the accompanying drawings.

[0086] like Figure 1-8 As shown, according to an embodiment of the present application, a method for preparing a monoclonal antibody against a recombinant P29 protein of Echinococcus multilocularis comprises the following steps:

[0087] S1. Animal husbandry: The experimental animals were 10 6-8 week old Balb / c female mice, all of which were housed in independent ventilated mouse cages with adequate light and suitable temperature. They were adaptively housed for one week before the experiment.

[0088] S2. Construction and purification of recombinant protein P29, wherein the steps include:

[0089] 1) Cultivation of rEg.P29 recombinant strain and induction of expression of P29 protein, the specific steps include:

[0090] ① Single colony culture: Take out the E. coli BL21 (DE3) plysS glycerol strain containing the Eg.P29 / pET28a recombinant expression plasmid stored in the laboratory from the -80℃ refrigerator, thaw at room temperature, operate aseptically in the clean bench, draw 20μL of bacterial solution on the four zones of LB solid medium (containing kanamycin). Invert and place in a 37℃ incubator for overnight incubation, and observe the formation of single colonies on the culture plate.

[0091] ② Induce expression: Transfer the bacterial solution to 10 mL LB liquid culture medium in two 50 mL sterile EP tubes at a ratio of 1:100, shake the culture in a shaker at 37°C and 200 rpm for 6 hours. When the bacterial solution becomes turbid and the OD600nm value is about 0.5-0.6, add IPTG inducer (final concentration 0.1 mM) to one of the tubes and continue shaking overnight to induce full protein expression.

[0092] ③ Collect bacteria and prepare samples. Pipette 1 mL of bacterial solution from two tubes into 1.5 mL centrifuge tubes, centrifuge at 12000 rpm for 5 minutes, discard the supernatant, add 100 μL of PBS and 25 μL of 5× protein loading buffer, mix by pipetting, and heat in a 100°C metal bath for 8 minutes to denature the protein.

[0093] ④SDS-PAGE detection of target protein, the specific steps include:

[0094] a. Preparation of lower separation gel: According to the instructions of the YAZY PAGE Gel Rapid Preparation Kit, pipette 3 mL of lower gel solution (2×) and lower gel buffer (2×) into a mixing cup, add 60 μL of coagulant to the separation gel solution, mix thoroughly and then slowly add to a 1 mm glass plate gel tank, flatten the upper layer with anhydrous ethanol, and let stand at room temperature for about 30 minutes to allow the lower separation gel to solidify.

[0095] b. Preparation of upper concentrated gel: discard the upper layer of anhydrous ethanol, absorb the excess alcohol with absorbent paper strips, and according to the instructions of the YAZY PAGE Gel Rapid Preparation Kit, pipette 1 mL each of the upper gel solution (2×) and the colored upper gel buffer (2×) into the mixing cup, add 20 μL of the coagulant to the concentrated gel solution, mix thoroughly and slowly add it into the glass plate gel tank. When it is almost full, insert a 15-hole comb with a thickness of 1 mm. After it solidifies at room temperature for about 1 hour, gently pull out the comb in the vertical direction.

[0096] c. Sample addition: Slowly add the prepared bacterial protein solution into the sample wells, 10 μL per well, and be careful not to let the sample leak into other wells.

[0097] d. Electrophoresis: Connect the power supply according to the positive and negative poles, set the constant voltage to 80V, and start electrophoresis. Observe the position of bromophenol blue, and when the sample runs out of the concentrated gel to the separation gel (about 30 minutes), adjust the voltage to 120V until the bromophenol blue runs to the bottom of the glass plate (about 1 hour), and stop electrophoresis.

[0098] e. Coomassie Brilliant Blue Staining: Use a special gel cutting board to carefully pry open the glass plate, peel the gel along the edge to the staining box, add an appropriate amount of Coomassie Brilliant Blue staining solution (to ensure that the gel is completely covered), and use a transfer decolorization shaker to slowly shake and stain for 30 minutes.

[0099] f. Decolorization: Recover the Coomassie Brilliant Blue solution, wash off the floating color with tap water, add decolorizing solution to cover the gel, and heat in a microwave oven to decolorize. Note that the process should be heated at a low temperature and observe while heating to prevent the liquid from boiling dry.

[0100] g. Take photos: After the decolorization is complete, the protein bands are visible to the naked eye. Use the protein imaging system to select the Protein Gels / Coomassie Blue mode to take photos. The detection of a clearly bold protein band of about 29KD in the IPTG-induced bacterial solution proves that the bacterial solution is successfully induced and the protein can be purified by shaking.

[0101] Test results see Figure 1 , where Lane 1: Marker; Lane 2: uninduced expression; Lane 3: 0.1 mM IPTG induction; Lane 4: purified rEm.P29 protein.

[0102] 2) Purification of rEgP29, the specific steps include:

[0103] ① Shake vigorously: Add the qualified bacterial solution into a conical flask containing 300 mL LB liquid culture medium at a ratio of 1:100. After shaking at 200 rpm at 37°C for 6 hours, when the OD600nm value is about 0.5-0.6, add IPTG inducer (final concentration 0.1 mM) and continue shaking overnight to induce protein expression.

[0104] ② Collect bacteria: collect the shaken bacterial liquid, balance it and centrifuge it at 6000rpm (4℃) for 30min in a high-speed centrifuge, collect the supernatant into a waste liquid bucket (discard it after high pressure), resuspend and wash the precipitated bacteria with PBS, centrifuge it at 6000rpm (4℃) for 30min, collect the precipitated bacteria and weigh them.

[0105] ③Bacterial lysis: Add one-tenth of the original bacterial solution volume of Binding Buffer to the precipitated bacterial cells to resuspend the bacterial cells, add 16μL lysozyme (5mg / mL) and 4μL PMSF (100mM) per gram of bacterial cells, mix well, and place on ice for 30 minutes. Ultrasonic cell pulverizer, parameter settings: Timer: 5min, Tem: 4℃, Pause on: 1s, Pause off: 1.5s, intensity 30%. Place the 50mL centrifuge tube containing the bacterial solution in an ice bath for ultrasonic lysis. Be careful not to let the ultrasonic metal rod touch the tube wall during the ultrasonic process. If you hear a sharp metal sound, pause immediately, adjust the position and continue to lyse the bacteria. Repeat 2 to 3 times, and stop ultrasonication when the bacterial solution becomes clear.

[0106] ④Purification: Centrifuge the fully lysed transparent bacterial solution at 12000rpm and 4℃ for 30min, collect the supernatant and mix it with the activated His-bind resin affinity chromatography column containing Ni2+, shake it at 4℃ overnight to allow the protein to fully bind to the nickel column. Filter the bacterial solution containing the nickel column to the column bed, and collect the filtrate to determine whether the protein is fully bound. Add 5 times the volume of the nickel column Washing buffer to wash twice, and collect the washing liquid to determine whether the washing liquid has eluted target protein. Add 5 times the volume of the nickel column Elution buffer, collect the slowly dripping eluate in a 1.5mL centrifuge tube, 1mL per tube, collect 10 tubes in total, and mark the serial number. The purified protein is placed on ice, the purity is detected by SDS electrophoresis, and the protein concentration is determined by the BCA method.

[0107] 3) Protein dialysis, the specific steps include:

[0108] ① Activation of dialysis bag: Select a dialysis bag with a molecular weight cutoff of 8000-14000KD, cut about 10cm of the dialysis bag with scissors, boil it in distilled water for 5-10 minutes, activate the dialysis bag and cool it in cold water.

[0109] ② Dialysis: Put the purified P29 protein eluted by Elutionbuffer into a dialysis bag, clamp both ends, place it in a large beaker filled with 2000mL pre-cooled PBS, add a magnetic stirrer and place it in a 4℃ refrigerator, stir and dialyze, the stirring speed should not be too fast. Change the dialysis solution every 4 hours, and dialyze overnight after changing it three times.

[0110] ③ Collect protein: After dialysis overnight, centrifuge the protein solution at 12000rpm for 10min, collect the protein supernatant, and measure the protein concentration.

[0111] 4) The protein concentration was determined by BCA method, and the specific steps included:

[0112] ① Prepare BCA working solution: Calculate the volume of the working solution according to the number of samples to be measured = (standard wells + number of samples to be measured) × 0.2 mL, and mix reagent A and reagent B at a ratio of 50:1.

[0113] ②Prepare standard pore protein: add standard protein solution and deionized water into the standard wells respectively, with a total volume of 20 μL, so that the protein content of the standard is 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 μg, respectively.

[0114] ③Sample dilution: After the protein to be tested is diluted 5 times, add 20 μL to the sample well.

[0115] ④ Add 200 μL / well of BCA working solution to the standard wells and sample wells respectively, mix well and incubate in a 37°C incubator for about 15 minutes.

[0116] ⑤OD value determination: When the standard wells are stained blue, measure the OD value of the 96-well plate at a wavelength of 562 nm on an ELISA reader.

[0117] ⑥ Draw a standard curve to calculate the protein concentration, and store the protein in a -80℃ refrigerator for later use.

[0118] Specifically, the operation was performed according to the instructions of the corresponding BCA protein concentration determination kit.

[0119] S3. Preparation and application of monoclonal antibodies, wherein the steps include:

[0120] 1) Immunization scheme: P29 protein from which imidazole was removed by dialysis was used to immunize 5 BALB / c mice, with a dose of 50 μg / time, once every week, for a total of three immunizations, with complete Freund's adjuvant used for the first immunization and incomplete Freund's adjuvant used for the next two immunizations.

[0121] 2) Immunization method: After the frozen recombinant P29 protein is thawed on ice, it is sterilized by filtering using a 0.22 μm filter, and the protein concentration is diluted to 500 μg / ml with Freund's adjuvant (complete or incomplete). After sufficient low-temperature emulsification, 100 μL of protein emulsifier is injected subcutaneously at three points in the abdomen of each mouse.

[0122] 3) Determination of serum antibody titer of immunized mice, the specific steps include:

[0123] ①Collect serum from the tail vein of mice: Collect blood from the tail vein of mice 2 weeks after the last immunization. After fixing the mouse by hand, wipe the end of the mouse tail with an alcohol cotton ball, cut off about 2 mm of the end of the mouse tail with sterile scissors, collect about 50 μL of blood from the broken end in a 1.5 mL centrifuge tube, and stop bleeding with electrocautery. Place the collected whole blood of mice at 4°C overnight, centrifuge at 3000g for 10 minutes, and draw the upper serum for later use.

[0124] ②The ELISA method was used to detect the P29-specific antibody titer in the sera of 5 mice.

[0125] a. Antigen coating: Design the coating plate according to the experimental needs and mark the strips. Dilute the P29 antigen to a concentration of 1 μg / mL with coating buffer solution (pH 7.4), mix well and add to the strips, 100 μL per well, and incubate in a 4°C refrigerator overnight.

[0126] b. Blocking: Discard the coating solution and wash the plate 5 times with PBST buffer, shaking for 3 minutes each time. After the last wash, shake off the PBST as much as possible and pat the remaining liquid on absorbent paper. Add 200μL of 5% skim milk diluted with PBST to each well for blocking and incubate in a 37℃ incubator for 2 hours.

[0127] c. Primary antibody dilution: Use 5% skim milk to dilute the collected mouse antiserum at a starting dilution of 1:500, and then dilute it 3-fold to 11 concentrations. The concentrations of the antiserum dilutions are: 1:500, 1:1500, 1:4500, 1:13500, 1:40500, 1:121500, 1:364500, 1:1093500, 1:3280500, 1:9841500, and 1:29524500.

[0128] d. Incubate with primary antibody: discard the blocking solution, wash the plate 5 times with PBST buffer, 3 min / time, shake off the washing solution as much as possible after the last wash, and pat dry the residual liquid on absorbent paper. Add 100 μL of diluted antiserum to each well, and normal mouse serum (concentration 1:1000) to the control well, and incubate at 37℃ incubator for 1 hour.

[0129] e. Incubate with secondary antibody: discard the primary antibody, wash the plate 5 times with PBST buffer, 3 min / time, shake off the washing solution as much as possible after the last wash, and pat dry the remaining liquid on absorbent paper. Dilute the anti-mouse antibody (HRP) with 5% skim milk at 1:10000, add 100 μL of the diluted secondary antibody to each well, and incubate in a 37°C incubator for 1 hour.

[0130] f. Color development: discard the secondary antibody, wash the plate 7 times with PBST buffer, 3 minutes each time, shake off the washing solution as much as possible after the last wash, and pat dry the remaining liquid on absorbent paper. Add 100μL TMB color development solution to each well, react at 37℃ in the dark for 10-15 minutes, and pay attention to observe the color of the liquid in the well gradually turning blue.

[0131] g. Stop color development: After the color develops to an appropriate color, add 50 μL of TMB color stop solution to each well to stop the color development. The color in the well will turn yellow.

[0132] h. Determine OD value: Measure the OD value of each well at a wavelength of 450 nm on an ELISA reader within 30 minutes after stopping the color development.

[0133] After mice were immunized four times with rEm.P29 combined with Freund's adjuvant, blood was collected from the tail vein to detect serum antibody titers. The results showed that the antiserum titer of mouse 1# was around 1093.5K, and the antiserum titers of mice 2#, 4#, and 5# were around 3280.5K. The antiserum titer of mouse 3# was 9841.5K. A fusion experiment was performed on mouse 3#, and the experimental results are shown in Figure 3 ;

[0134] Among them, A.1# mouse serum antibody titer; B.2# mouse serum antibody titer; C.3# mouse serum antibody titer; D.4# mouse serum antibody titer; E.5# mouse serum antibody titer.

[0135] S4, hybridoma cell preparation, wherein the steps include:

[0136] 1) Preparation of myeloma cells: One week before fusion, SP 2 / 0 cells were expanded and cultured in DMEM medium containing 10% FBS. At the time of fusion, the cells filled about 6 T25 cell culture flasks. On the day of fusion, SP 2 / 0 cells were collected into a 50 mL centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and then 20 mL of DMEM basal medium was added. The cells were mixed by pipetting and counted.

[0137] 2) Preparation of spleen cells: After the third immunization, the mice with the highest serum antibody titer detected by ELISA were selected as fusion objects. On the day of fusion, the mice were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes. The spleen was taken out by sterile operation and placed in a 10cm culture dish containing 15mL DMEM basal culture. The sieve was placed in another culture dish, and the spleen was transferred to a 70μm cell sieve. The spleen was gently ground with a 5mL syringe core rubber head. The sample diluent was added to the sieve while grinding, and the sieve was rinsed to collect more spleen cells in a 50mL centrifuge tube, with a final volume of about 5mL. 5mL of mouse lymphocyte separation solution was added to a 15mL sterile centrifuge tube, and then the spleen cell suspension obtained by grinding was spread on the lymphocyte separation solution, and centrifuged at 450g (slow rise and slow fall or rise 7 and fall 0) for 20min. The supernatant was discarded, and the middle white film layer was collected into a new 15mL centrifuge tube, 10mL of washing solution was added, and centrifuged at 350g for 8min at room temperature. Discard the supernatant, add 5 mL of DMEM basal medium, mix by pipetting, and count for later use.

[0138] 3) Cell fusion: Add 1×107 myeloma cells to 2×107 spleen cell suspension and mix well, so that the ratio of myeloma cells to spleen cells is preferably 1:2. Place the cells in a 50mL centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant, wash the cells once with 10mL of electrofusion solution, then centrifuge at 1000rpm for 5min, and discard the supernatant. Dilute the cells to 2×107 / mL with electrofusion solution, then add 2mL of cell suspension to the fusion pool for electrofusion experiment, and let it stand for 10min after electroporation. Then add HAT (Hypoxanthine, Aminopterin, Thymidine) culture medium to 10mL and mix well by blowing. Spread the fused cells into a 96-well plate, 100μL per well. Then place the cell culture plate in a CO2 incubator for culture. Check the hybridoma cell clone status and cell growth status 6 days after fusion, and perform screening tests 10 days after fusion.

[0139] 4) Screening of fused cells, the specific steps include:

[0140] ① Antigen coating: Design the coating plate according to the experimental needs and mark the strips. Dilute the P29 antigen to a concentration of 1 μg / mL with coating buffer solution (pH 7.4), mix well and add to the strips, 100 μL per well, and store in a 4°C refrigerator overnight.

[0141] ② Blocking: discard the coating solution, wash the plate 5 times with PBST buffer, 3 minutes each time. After the last wash, shake off the PBST as much as possible, pat dry the remaining liquid on absorbent paper, add 200μL PBST diluted with 5% skim milk to each well for blocking, and incubate at 37℃ for 2 hours.

[0142] ③ Incubate with primary antibody: discard the blocking solution, wash the plate 5 times with PBST buffer, 3 min / time, shake off the washing solution as much as possible after the last wash, and pat dry the residual liquid on absorbent paper. Add 100 μL of hybridoma cell supernatant to each well, and normal mouse serum (concentration 1:1000) to the control well, and incubate in a 37°C incubator for 1 hour.

[0143] ④ Incubate with secondary antibody: color development, color termination, and OD value determination methods are the same as 2.2.3 mouse serum antibody titer determination.

[0144] Among them, the positive wells are judged as follows: the sample well OD value / negative well OD value ≥ 2.1, then it is judged as a positive well. Use a single-channel pipette to pick the positive wells detected on the whole plate, and perform a second confirmation test to further confirm the positive wells. The confirmed positive wells are subcloned.

[0145] Select the 3# mouse with the highest mouse antiserum antibody titer, isolate spleen cells, and electro-fuse with myeloma cells SP2 / 0. After dilution, the fused cells were spread on 4 96-well plates. After 10 days of culture, the culture supernatant was tested for anti-P29 antibodies by ELISA. The results are shown in Table 1, Table 2, Table 3, and Table 4. A total of 5 fusion-positive cell lines with specific reactions to Em.P29 protein were obtained, including 1F3, 2G6, 2H4, 3F9, and 4F11. Red represents positive wells.

[0146] Table 1 Plate fusion cell screening results

[0147]

[0148] Table 2 Plate fusion cell screening results

[0149]

[0150] Table 3 Plate fusion cell screening results

[0151]

[0152] Table 4 Plate fusion cell screening results

[0153]

[0154] The culture supernatants of the positive wells 1F3, 2G6, 2H4, 3F9, and 4F11 screened out above were collected and diluted at 1:1, 1:10, 1:100, 1:1000, and 1:10000. The antibody titers in the culture supernatants were detected by ELISA. The experimental results are as follows: Figure 4 .

[0155] 5) Cell subcloning and subclone screening, the specific steps include:

[0156] ① Cell subcloning: Subclone hybridoma cells by limiting dilution method, the method is as follows:

[0157] Blow up the cells in the positive wells and count them as N. Add N / 4mL DMEM medium to the centrifuge tube, take 100μL of cell suspension into the centrifuge tube, blow evenly and leave 1mL, add DMEM to 4mL, blow evenly, and leave 100μL (about 2 drops) at the bottom of the tube. Add DMEM to 5mL in the centrifuge tube, mix well and add to the first three rows of the 96-well plate, 100μL per well. Leave about 1.8-2mL at the bottom of the tube, add DMEM to 5mL, blow evenly and add dropwise to rows D, E, and F of the 96-well plate, 100μL for each well. Leave about 1.5-1.8mL at the bottom of the tube, add DMEM to 2.8-3mL, blow evenly and add dropwise to rows G and H of the 96-well plate, 100μL for each well. Observe under a microscope after 7-10 days, detect the wells with single clone growth, mark the monoclonal wells, pick the positive monoclonal cells for subcloning again, and after detecting 100% positivity, pick the monoclonal wells for expansion culture and finalization.

[0158] ②Subclone screening: The method is the same as fusion cell screening.

[0159] The limiting dilution method was used to perform subclone screening with the positive wells screened above. The culture supernatant was collected by culturing the diluted cells, and the changes in specific antibodies in the culture supernatant were detected by ELISA. The results showed that a total of 74 monoclonal cell lines were subcloned, of which 22 were positive wells, as shown in Table 5, and red indicated positive cell lines. The 22 positive cell lines screened in Table 5 were verified again, and combined with cell viability, 4 positive cell lines were finally screened out, and the results are shown in Table 6.

[0160] Table 5 Subclone screening results

[0161]

[0162] Table 6 Subclone screening results verified again

[0163]

[0164] After the positive cell lines screened by subcloning were graded diluted, the production of anti-Em.P29 specific antibodies in the culture supernatant was detected by ELISA. Figure 5 shown.

[0165] S5. Application of monoclonal antibodies, wherein the steps include:

[0166] 1) Determination of monoclonal antibody cell line subtypes: Use indirect ELISA to detect the monoclonal antibody subtypes produced by the screened positive monoclonal cell lines. Antigen coating, plate washing, blocking, and primary antibody incubation methods are similar to those for fusion cell screening. HRP-labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgM are used as secondary antibodies. Color development, termination, and OD value detection are the same as before.

[0167] 2) Determination of monoclonal antibody titer: Collect the culture supernatant of the selected myeloma cell lines, use the BCA method to detect the protein concentration in the culture supernatant, and adjust the culture supernatant protein concentration to be consistent. Use the indirect ELISA method to detect the antibody titer in the culture supernatant of each cell line, the method is the same as the mouse serum antibody titer determination in S3.

[0168] 3) Purification of monoclonal antibodies: The selected monoclonal antibody cell lines were expanded without serum and the culture supernatant was collected. The protein G agarose gel medium was loaded into the affinity purification chromatography column, and the cell culture supernatant was slowly loaded. After the antibody was fully bound, it was eluted with glycine elution buffer to obtain the desired purified antibody, which was immediately dialyzed in PBS at 4°C overnight. The purified antibody was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining to detect the purity of the antibody. The concentration of the antibody was detected using the BCA kit, and the antibody titer was detected using the ELISA method.

[0169] The monoclonal cell line was expanded and the anti-Em.P29 specific antibody was purified from the culture supernatant using protein G. The antibody titers of the four monoclonal antibodies were tested by ELISA. The results showed that among the four monoclonal antibodies, the antibody titer of the 1G6F7 monoclonal cell line was the highest, followed by 1F3D4. Figure 6 shown.

[0170] 4) Evaluation of monoclonal antibodies on the infection model prepared by intraperitoneal injection of alveolar protoscolex in mice. The specific results are shown in Figure 6 .

[0171] Among them, the evaluation of monoclonal antibodies on the infection model prepared by intraperitoneal injection of alveolar protoscolex in mice can be divided into:

[0172] ① Isolation and activity detection of alveolar protoscolex;

[0173] ② Preparation of the mouse intraperitoneal injection model of alveolar protoscolex infection. The specific operation is as follows: isolate the protoscolex with good vitality, resuspend it with sterile PBS to a concentration of 10,000 heads / mL, and use a 1mL empty needle to draw 200μL of protoscolex suspension (2,000 heads) and inject it into the left lower abdomen of the mouse. Note that because the protoscolex sediments quickly, the protoscolex suspension should be drawn once for each mouse injected, and mixed while drawing. The syringe and centrifuge tube after injection should be discarded after high pressure;

[0174] ③ Grouping and intervention: Specific operations were as follows: 12 weeks after infection, the mice underwent abdominal B-ultrasound examination to examine the formation of abdominal vesicular infection foci, and the mice with successful infection were selected and randomly divided into 5 groups, with 9 mice in each group.

[0175] This experiment set up three P29 monoclonal antibody dose groups: low, medium, and high, to screen the optimal dose of the monoclonal antibody, and used the isotype IgG1 monoclonal antibody as the control group.

[0176] a. Isotype control group: 200 μL (5 mg / kg) of mouse isotype IgG1 monoclonal antibody was injected into the tail vein once a week;

[0177] b. P29 monoclonal antibody group (2.5 mg / kg): 200 μL (0.25 mg / mL) of P29 monoclonal antibody was injected into the tail vein once a week;

[0178] c. P29 monoclonal antibody group (5 mg / kg): 200 μL (0.5 mg / mL) of P29 monoclonal antibody was injected into the tail vein once a week;

[0179] d. P29 monoclonal antibody group (10 mg / kg): 200 μL (1 mg / mL) of P29 monoclonal antibody was injected into the tail vein once a week;

[0180] e. ABZ group: ABZ was administered intragastrically at 50 mg / kg (5 mg / mL, 200 μL) once a day. The mice were weighed and recorded once a week.

[0181] After 8 weeks of intervention, samples were collected for evaluation. The cysts were weighed to calculate the cyst inhibition rate; the liver and spleen weights of the mice were weighed as well as the body weight of the mice to calculate the liver index and spleen index.

[0182] 5) Evaluation of 1G6F7 monoclonal antibody on the infection model prepared by portal vein injection of alveolar protoscolex in mice. The specific results are shown in Figure 7 .

[0183] Among them, the evaluation of 1G6F7 monoclonal antibody on the infection model prepared by portal vein injection of alveolar protoscolex in mice can be divided into:

[0184] ① Isolation and activity detection of alveolar protoscolex

[0185] ② Preparation of the mouse portal vein injection model of alveolar protoscolex infection, the specific operation is as follows: isolate the protoscolex with good vitality, and resuspend it in sterile PBS to a concentration of 10,000 heads / mL. The mouse abdomen was depilated and skinned. After satisfactory anesthesia, the mouse was fixed in a supine position on the operating table. The abdomen was disinfected with iodine 3 times. An incision of about 1 cm below the xiphoid process was made with ophthalmic scissors. A 4 cm sterile gauze was placed around the incision and fixed with adhesive tape. The small intestine was gently pulled out with blunt curved forceps until the portal vein was exposed. 100 μL of protoscolex suspension (1000 heads) was drawn with a 1 mL empty needle and slowly injected along the portal vein. After the injection, a cotton swab was used to press and stop bleeding immediately, and hemostatic powder was filled locally until the incision was sutured immediately after the bleeding stopped. The postoperative mice were placed in a 37 ° C incubator for warming and rewarming. The mice in the sham operation group were injected with an equal volume of PBS solution. After the mice woke up, they were transferred to the breeding cage and the life activities of the mice were closely monitored. The instruments, needles, and pillows after the operation were all treated with high pressure.

[0186] ③ Grouping and intervention: The specific operation is as follows: Due to the difficulty of portal vein infection surgery, intraoperative hemostasis is difficult, and injection of protoscolex can easily cause portal vein thrombosis and infection, the surgical mortality rate of mice is high, and the number of model mice prepared is small. The surviving mice after surgery were randomly divided into 2 groups, 5 in each group. Intervention began 1 week after infection.

[0187] a. Albendazole group: intragastrically administered with 200 μL of abendazole solution (5 mg / kg), once a week;

[0188] b. P29 monoclonal antibody group (5 mg / kg): 200 μL (0.5 mg / mL) of P29 monoclonal antibody was injected into the tail vein once a week;

[0189] c. Sham operation group: 200 μL PBS was injected into the tail vein once a week.

[0190] The ABZ group was not set up in this experiment due to the small number of mice surviving. The mice were weighed and recorded once a week.

[0191] The results of the evaluation of the therapeutic effect of 1G6F7 monoclonal antibody on the mouse model of intraperitoneal infection of Echinococcus multilocularis are shown in Figure 7 , among which, A. changes in cyst weight; B. changes in liver index; C. changes in spleen index.

[0192] The results of the evaluation of the therapeutic effect of 1G6F7 monoclonal antibody on the mouse model of Echinococcus multilocularis hepatic portal vein infection are shown in Figure 8 , among which, A. changes in liver index; B. changes in spleen index.

[0193] The above statistical results were analyzed and processed using GraphPadPrism software 8.0, and the data were expressed as mean ± standard deviation. Student's t test was used for two groups of data, and one-way analysis of variance was used for multiple groups of data. p<0.05 indicated statistically significant differences.

[0194] The above are only embodiments of the present application and are not intended to limit the scope of protection of the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present application. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings.

[0195] The above are only specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for preparing a monoclonal antibody against recombinant P29 protein of Echinococcus multilocularis, characterized in that: The following steps are involved: S1. Animal husbandry: The experimental animals were 10 6-8 week old female Balb / c mice; S2. Construction and purification of recombinant protein P29, wherein the steps include: 1) Cultivation of rEg.P29 recombinant strain and induced expression of P29 protein; 2) Purification of rEgP29; 3) Protein dialysis; 4) Determination of protein concentration by BCA method; S3. Preparation and application of monoclonal antibodies, wherein the steps include: 1) Immunization regimen; 2) Immunization method; 3) Determination of serum antibody titer of immunized mice; S4, hybridoma cell preparation, wherein the steps include: 1) Myeloma cell preparation; 2) Preparation of spleen cells; 3) Cell fusion; 4) Screening of fusion cells; 5) Cell subcloning and subclone screening; S5. Application of monoclonal antibodies, wherein the steps include: 1) Monoclonal antibody cell line subtype determination; 2) Determination of monoclonal antibody titer; 3) Purification of monoclonal antibodies; 4) Evaluation of monoclonal antibodies on the infection model prepared by intraperitoneal injection of alveolar protoscolex in mice; 5) Evaluation of 1G6F7 monoclonal antibody on the infection model prepared by portal vein injection of alveolar protoscolex in mice.

2. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S2, the specific steps of culturing the rEg.P29 recombinant strain and inducing expression of the P29 protein include: S1, single colony culture; S2, induced expression; S3, collect bacteria and prepare samples; S4. SDS-PAGE detection of target protein. The specific steps include: 1) Preparation of lower separation gel; 2) Preparation of upper concentrated gel; 3) Add sample; 4) Electrophoresis; 5) Coomassie Brilliant Blue staining; 6) Decolorization; 7) Take photos.

3. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S2, the specific steps of purifying rEgP29 include: S1, big shake; S2, collecting bacteria; S3, schizobacterium; S4. Purification.

4. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S2, the specific steps of protein dialysis include: S1, dialysis bag activation; S2, dialysis; S3. Collect protein.

5. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S2, the specific steps of determining the protein concentration by BCA method include: S1. Prepare BCA working solution; S2, prepare standard porins; S3, sample dilution; Add 200 μL / well of BCA working solution to S4, standard wells and sample wells, mix well and incubate at 37°C for about 15 min. S5. OD value determination; S6. Draw a standard curve to calculate the protein concentration, and store the protein in aliquots at -80°C for later use.

6. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S3, the specific steps of determining the antibody titer of the immunized mouse serum include: S1, collecting serum from the tail vein of mice; S2. The ELISA method was used to detect the P29-specific antibody titer in the sera of 5 mice.

7. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In the above S5, the hybridoma cells are subcloned by limiting dilution method, and after detection to 100% positive, single clone wells are picked out for expansion culture and determination of strains for subclone screening.

8. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S5, both fusion cell screening and subclone screening are screened and detected by the indirect ELISA method.

9. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S5, monoclonal antibodies are purified using Protein G agarose gel medium and tested, and an infection model is established by injecting alveolar protoscolex into the peritoneal cavity or portal vein of mice.

10. The method for preparing the monoclonal antibody against the recombinant P29 protein of Echinococcus multilocularis according to claim 1, characterized in that: In S5, the evaluation steps of the infection model prepared by intraperitoneal injection of the monoclonal antibody to mice and the infection model prepared by portal vein injection of the monoclonal antibody 1G6F7 to mice are: S1. Isolation and activity detection of alveolar protoscolex, the method is the same as the determination of serum antibody titer of immunized mice; S2. Preparation of mouse intraperitoneal injection of alveolar protoscolex infection model; S3. Grouping and intervention.

Images