CN113045653A - Monoclonal antibody for inhibiting plasmodium infection and preparation method and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
- C07K16/205—Plasmodium
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- A—HUMAN NECESSITIES
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- A61K39/39575—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from other living beings excluding bacteria and viruses, e.g. protozoa, fungi, plants
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- A—HUMAN NECESSITIES
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- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
- A61K2039/507—Comprising a combination of two or more separate antibodies
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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Abstract
The invention relates to the technical field of animal cells, and particularly discloses a preparation method of a monoclonal antibody for inhibiting plasmodium infection, which specifically comprises the following steps: obtaining positive clone, prokaryotic expression, purifying recombinant protein, immunizing a mouse by the recombinant protein, preparing fusion cells, collecting spleen cells of the immunized mouse, fusion culture, monoclonal screening and culturing hybridoma cells. The monoclonal antibodies PfRH5-1 and PfRH5-2 obtained by the invention can obviously inhibit plasmodium from invading erythrocytes and reduce the infection rate of plasmodium.
Description
Technical Field
The invention relates to the technical field of animal cells, in particular to a monoclonal antibody for inhibiting plasmodium infection and a preparation method and application thereof.
Background
Malaria is an important parasitic disease transmitted by the blood-sucking of female anopheles bites. WHO '2020 world malaria report' states that: there were 2.29 billion cases of malaria infection in 91 countries in 2019, with a global total number of malaria deaths of 40.9 million people, and the data indicates no slowing trend in the number of malaria patients worldwide over the last four years. Plasmodium is the causative agent of malaria, and there are 5 species of plasmodium infecting humans: including plasmodium falciparum, plasmodium vivax, plasmodium malariae, plasmodium ovale and plasmodium knowlesi, plasmodium falciparum is the most prevalent plasmodium in african regions, accounting for 99.7% of 2019 cases of malaria. The life history of plasmodium includes both the development in humans and the development in female anopheles mosquitoes. The development in the human body includes two stages, an infrared stage and a red phase. By infecting a human body by biting female anopheles mosquito carrying plasmodium, plasmodium sporozoites enter the human body, are released into blood after being developed and proliferated in the liver, are partially phagocytized by phagocytes, and partially invade erythrocyte to be proliferated by schizonts, so that a great amount of damage of the erythrocyte causes fever, anemia, splenomegaly, kidney damage and the like. In areas with high prevalence of malignant malaria, children under 5 years old, pregnant women, AIDS patients and people without malaria immunity are easy to cause dangerous malaria, and if the children are not treated in time or treated improperly, the patients are easy to die. Although malaria remains a serious public health problem with high morbidity and mortality worldwide after years of control, it is also known as the world's three major infectious diseases along with tuberculosis and aids.
Antimalarial drugs, mosquito-vector control and malaria vaccines are important means for the control of malaria, however, drug-resistant strains of plasmodium and anopheles have emerged worldwide, rendering malaria control a serious challenge. The invasion of erythrocytes by merozoites of the plasmodium, which are mediated by the interaction between merozoite ligands and host receptors, is a key mechanism for the pathogenesis of malaria. Plasmodium merozoites utilize multiple ligands, such as: the Erythrocyte-Binding protein (EBL) family and Reticulocyte Binding protein (RBL) family invade erythrocytes. PfRH5 is a new member of the Plasmodium falciparum RBL family, is located in the rod-shaped body at the top of merozoites and is an important protein which is discovered in recent research and is involved in merozoite invasion. The significance of the discovery is that the combination of PfRH5 and a ligand thereof exists in a plurality of strains of plasmodium falciparum, and the previously discovered ways capable of mediating the invasion of erythrocytes by plasmodium are only effective for a few strains of plasmodium falciparum; it has also been shown that PfRH5 based vaccines are able to develop resistance against different species of plasmodium in primate nocturnal monkeys. Therefore, PfRH5 is honored as the master key for opening the Ashi's heel of Plasmodium falciparum.
Currently, no monoclonal antibody against plasmodium falciparum RH5 has been used to inhibit plasmodium merozoite invasion. Since polyclonal antibodies are complex in antibody composition and are not suitable for clinical treatment, it is necessary to develop effective monoclonal antibodies.
Disclosure of Invention
In order to solve the technical problems, the invention provides the monoclonal antibody for inhibiting plasmodium infection and the preparation method and the application thereof, and the two prepared monoclonal antibodies can be used for detecting plasmodium falciparum or screening corresponding medicines.
The first purpose of the invention is to provide a preparation method of a monoclonal antibody for inhibiting plasmodium infection, which comprises the following steps:
s1, acquisition of positive clones: synthesizing and cloning coding genes corresponding to the amino acid sequences of the sections 201 and 324aa of the PfRH5 protein sequence into a prokaryotic expression vector PGEX4T-1, selecting a positive cloning marker as PfRH5-1, synthesizing and cloning coding genes corresponding to the amino acid sequences of the sections 302 and 396aa of the PfRH5 protein sequence into the prokaryotic expression vector PGEX4T-1, and selecting a positive cloning marker as PfRH 5-2;
s2, prokaryotic expression:
respectively inoculating the two positive clone strains into an LB culture medium containing antibiotics for culture, adding an inducer IPTG (isopropyl-beta-thiogalactoside) for continuous culture overnight, crushing escherichia coli, centrifuging the crushed bacterial liquid, and respectively collecting supernatant and precipitate;
s3, purification of recombinant protein:
filtering the collected supernatant with a 0.22-micron filter membrane, passing through a column, collecting target proteins PfRH5-1-GST and PfRH5-2-GST, eluting, dialyzing, and concentrating to 1mg/mL to obtain the antigen;
s4, immunization of mice: continuously carrying out three times of immunization, wherein each time is 22 days, 10 days after the third time of immunization, detecting the tail tip of an immune mouse to take blood, selecting a mouse with higher antibody titer to carry out boosting immunization, wherein the dosage of boosting immunization antigen is 48-50 mu g/mouse, tail vein injection is carried out, and 180-200 mu l of blood is used;
s5, preparation of fused cells: taking a normal mouse, and preparing a spleen cell suspension for culturing;
s6, collecting the splenocytes from the immunized mice: obtaining immune mouse splenocytes, adding erythrocyte lysate into cell sediment for resuspension and mixing evenly, and counting after lysing erythrocytes;
s7, fusion culture and monoclonal screening;
s8, culturing the hybridoma to obtain the monoclonal antibodies PfRH5-1 and PfRH 5-2.
Further, in S2, the culture conditions of the two positive clone strains are both 37 ℃, 250rpm, and the final concentration of the antibiotic in the LB medium is 100 g/mL.
Further, in S3, the dialysate was PBS, and the dialysis time was 12 hours.
Further, in S4, the three immunizations are specifically: the first immune antigen amount is 100 mu g/mouse, and the subcutaneous injection is performed at multiple points, and the total amount is 200 mu l; the 2 nd to 3 rd immunization, the antigen amount is 100 mug/mouse, and the subcutaneous multi-point injection is performed for 200 mul.
Further, in S5, the cell density was adjusted to 1X 10 in the spleen cell suspension culture5Adding into 96-well plate (100 μ l/well) at 37 deg.C with 5% CO2Culturing in an incubator.
Further, in S7, the specific process of fusion culture and monoclonal screening is as follows: get 108Cells were prepared for fusion as described for SP2/0 cells: mixing splenocytes at a ratio of 1:10, centrifuging to obtain cell precipitate, preheating, sucking 1ml of PEG preheated to 37 deg.C, adding into the cell precipitate, mixing, standing in water bath at 37 deg.C for 60s, adding HAT culture medium to terminate reaction, and adding HAT culture medium containing fused cellsInoculating to 96-well culture plate containing feeder layer cells, 100 μ l/well, 37 deg.C, 5% CO2Culturing in an incubator, performing monoclonal screening when the density of the culture medium occupies the holes 1/3, and selecting high-titer fusion positive holes.
Further, in S8, the specific process of culturing the hybridoma cell is as follows: selecting 10-week-old mice, inoculating incomplete adjuvant into abdominal cavity, wherein each mouse has an amount of 0.5ml, and injecting hybridoma cells diluted with serum-free culture medium into abdominal cavity, wherein the number of the injected cells is 2-3x106And collecting ascites after 7-10 days, centrifuging, collecting supernatant, and purifying to obtain monoclonal antibodies PfRH5-1 and PfRH 5-2.
The invention also provides the monoclonal antibody prepared by the preparation method, wherein the monoclonal antibody comprises an antibody PfRH5-1 and an antibody PfRH 5-2.
The invention also provides application of the monoclonal antibody in preparing a medicament for inhibiting or detecting plasmodium falciparum infection.
Further, the subject of the infection is red blood cells.
Compared with the prior art, the invention has the beneficial effects that:
1. the two high-specificity monoclonal antibodies are respectively PfRH5-1 and PfRH5-2, and both the two monoclonal antibodies PfRH5-1 and PfRH5-2 can obviously inhibit plasmodium from invading erythrocytes and reduce the infection rate of plasmodium;
2. the monoclonal antibody PfRH5-1 or PfRH5-2 prepared by the invention can be used for detecting plasmodium falciparum or screening corresponding drugs, and is the basis for clinically preventing or treating plasmodium falciparum related diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram showing the identification of the two monoclonal antibodies Westermblot obtained in example 1 of the present invention;
FIG. 2 is a graph showing the results of the detection of the two monoclonal antibodies against Plasmodium invasion erythrocytes obtained in example 1 of the present invention;
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified.
Example 1
This example provides a method for preparing monoclonal antibodies that inhibit infection by plasmodium, comprising the steps of:
s1, acquisition of positive clones: firstly, predicting a PfRH 5B cell epitope by jointly applying a Kyte-Doolittle method, an Emini method and a Jameson-Wolf method with the aid of a Gamier-Robson method, a Chou-Fasman method and a Karplus-Schulz method, preliminarily determining that a B cell epitope enrichment region is positioned in a 200-fold 400-aa section of a PfRH5 protein sequence, selecting coding genes corresponding to two sections of 201-fold 324-aa and 302-fold 396-aa amino acid sequences, synthesizing and cloning into a T vector, then cloning into a prokaryotic expression vector PGEX4T-1 respectively after correct sequencing analysis, and selecting positive clones which are respectively marked as PfRH5-1 and PfRH 5-2;
s2, prokaryotic expression:
two positive clones with correct sequencing result were inoculated into 5mL liquid LB medium containing antibiotic (final concentration of 100g/mL), cultured overnight at 37 ℃ at 250rpm, the overnight culture broth was transferred to 150mL liquid LB containing the above antibiotic at a ratio of 1:1000, culturing at 37 ℃ and 250rpm in a mass mode until OD600 is about 0.6, adding an inducer IPTG into a culture solution until the final concentration is 1mmol/L, continuing shaking culture overnight, centrifuging to obtain thallus precipitates, suspending the thallus in PBS (pH7.0) buffer solution, centrifuging the suspended thallus solution (4 ℃, 12000g and 5min), discarding the supernatant, repeating twice, ultrasonically crushing escherichia coli under ice bath conditions, centrifuging the ultrasonically crushed bacteria solution (4 ℃, 12000g and 15min), respectively collecting the supernatant and the precipitates, and detecting PfRH5-1-GST and PfRH5-2-GST proteins in the supernatant by SDS-PAGE;
s3, purification of recombinant protein:
purification of PfRH5-1-GST and PfRH5-2-GST was performed: washing the column with PBS for 4 times after filtering, centrifuging the ultrasonically crushed bacterial liquid (4 ℃, 12000g, 15min), collecting supernatant, filtering with a 0.22 mu m filter membrane, passing through the column, repeatedly passing through the column for 3 times, washing the column with PBS after filtering twice, washing off proteins, impurities and bound unfixed labeled non-target proteins which are not hung on the column, eluting proteins eluted by Buffer (L-reduced glutathione solution), collecting target proteins PfRH5-1-GST and PfRH5-2-GST, dialyzing the eluted proteins, changing PBS once every two hours until the overnight, and concentrating the dialyzed target proteins (antigens) to 1mg/mL by an ultrafiltration tube;
s4, recombinant protein immunization of mice: three times of immunization are carried out continuously, each time is 22 days apart, the amount of the primary immunization antigen is 100 mug/mouse, the immunization mode is that the subcutaneous multipoint injection of the Jiafu complete adjuvant is carried out, the total amount is 200 mug, the second immunization, the third immunization is carried out, 100 mug/mouse is carried out, the subcutaneous multipoint injection of the Jiafu incomplete adjuvant is carried out, the total amount is 200 mug, 10 days after the third immunization, the tail tip of the mouse with high antibody effect of the immunized mouse is detected for blood collection, the mouse with high antibody titer is selected for strengthening immunization, the amount of the strengthened immunization antigen is 50 mug/mouse, the tail vein injection is carried out, and the 200 mug;
s5, preparation of fused cells: taking a normal BALB/C mouse, removing an eyeball to collect blood, immersing the mouse in 75% alcohol solution after cervical dislocation and dying, and disinfecting for 5 minutes. Fixing on a dissecting table, cutting the abdominal skin with sterile scissors, exposing peritoneum, wiping the peritoneum with 75% alcohol cotton ball, replacing with a large-size needle with a 5ml syringe, first pumping 5ml of precooled RPMI-1640 basic culture medium, lifting the abdominal wall with forceps, injecting from one side of the abdomen to the abdominal cavity, massaging the abdomen for 1min to form cell suspension, pulling the side abdomen with forceps to form an abdominal sac, collecting the cell suspension, and pumping out the suspension with the syringe. Repeating the process for 2 times, transferring all into a pre-cooled 15ml centrifuge tube, centrifuging for 5min at 230g, discarding supernatant, resuspending with HAT medium, and counting cellsThereafter, the cells were adjusted to 1X 105Adding into 96-well plate, 100 μ l/well, culturing at 37 deg.C in 5% CO2 incubator;
s6, collecting the splenocytes from the immunized mice: blood is taken from eyeballs of the immunized mice, and serum is collected for standby after the blood is condensed. After the mice die at the dislocation of cervical vertebrae, putting the mice into 75% alcohol for disinfection for 5 min; the abdominal skin was cut open, the spleen was removed, the connective tissue outside the spleen was removed with forceps, and washed clean with PBS. The spleen was placed on a 200 mesh stainless steel cell screen and squeezed with the stylet of a 20ml syringe to allow the dispersed spleen cells to fall through the screen into a petri dish containing incomplete medium underneath. The screen is washed with a small amount of incomplete medium, the spleen cells are blown evenly, collected and centrifuged at 230g for 5 min. Discarding the supernatant, adding erythrocyte lysate into the cell sediment, resuspending and mixing, lysing erythrocytes according to cell sediment/lysate (V/V) ═ 1/2, standing for 3min, and centrifuging for 10min at 150 g. Removing supernatant, resuspending with 10ml of RPMI-1640 incomplete medium, centrifuging at 150g for 10min, discarding supernatant, resuspending with 10ml of RPMI-1640 incomplete medium, and counting a small amount of suspension.
S7, fusion culture and monoclonal screening: 108 cells were taken and used for fusion. Taking SP2/0 cells, using 10ml RPMI-1640 incomplete medium to resuspend, centrifuging, discarding supernatant, using RPMI-1640 incomplete medium to resuspend, and counting. According to SP 2/0: taking an appropriate amount of SP2/0 suspension from splenocytes at a ratio of 1:5 or 1:10, mixing the prepared splenocytes suspension and SP2/0 suspension, centrifuging for 5min at 90 g. The supernatant was aspirated off as thoroughly as possible, the bottom of the centrifuge tube was flicked slightly to loosen the cell pellet, and the pellet was preheated in a 37 ℃ water bath for use. Sucking 1ml of PEG preheated to 37 ℃, dropwise adding the PEG into the cell sediment within 60-90 s (slowly and quickly), stirring gently while adding, mixing uniformly, inclining a 50ml centrifuge tube to ensure that the PEG is fully contacted and uniformly mixed with the cells, and standing in a water bath at 37 ℃ for 60 s. HAT medium was added to terminate the reaction, and HAT medium containing the fused cells was inoculated into a 96-well plate containing feeder cells at 100. mu.l/well and cultured in a 5% CO2 incubator at 37 ℃. During the period, the liquid is changed according to the cell state, and when the state is good and the density of the liquid occupies 1/4-1/3, the monoclonal screening can be carried out. Selecting high titer fusion positive wells, transferring one part to 24-well plate, counting the rest, diluting to 1.5 and 1 cell per wellPlates were plated at 100. mu.l per well, with half of a 96 well plate per density. After 7 days, the single and multiple clones were identified by microscopic examination and marked. Measuring the titer of the monoclonal antibody by an ELISA method, calculating the positive porosity, selecting 5-10 wells with high titer (each well is monoclonal) per plate to transfer the wells to a 24-well plate if the titer is 100%, carrying out amplification culture to the 24-well plate, carrying out passage amplification to 3 wells, and carrying out primary cell cryopreservation when the density of each well is 80%. Gently blow up the cells from the well plate, blow evenly, and aspirate a small amount of cell suspension for cell counting. Calculating cell density and activity, and freezing and storing requirements: vigor > 90% and density 1X 106-1×107And/ml. And (3) sucking the cell suspension into a 15ml centrifuge tube, centrifuging for 3min at 90g, pouring out cell supernatant after centrifugation, adding precooled cryopreservation liquid with corresponding volume, lightly blowing and uniformly beating, and subpackaging in cryopreservation tubes of 1 ml/tube. Marking on the freezing tube: cell name, generation, density, date, operator name.
S8, hybridoma cell culture: BALB/C mice of about 10 weeks old are selected, and are inoculated with incomplete adjuvant in the abdominal cavity, and each mouse is 0.5 ml. The hybridoma cells diluted by serum-free culture medium are injected into the abdominal cavity, and the number of the injected cells is about 2x106A/only. After 7 days, observing the ascites generation condition of the mice every day, if the abdomen is obviously enlarged and the skin is tense when touching, collecting the ascites, centrifuging the ascites, and collecting the supernatant. The ascites fluid is purified.
The plasmodium lysate is taken as an antigen, western blot detection shows that the obtained monoclonal antibody has better specificity (shown in figure 1), and the titers of the obtained two monoclonal antibodies PfRH5-1 and PfRH5-2 are respectively: more than 1: 2000.
Monoclonal antibodies inhibit plasmodium proliferation: plasmodium falciparum (strain 3D 7) was treated synchronously with sorbitol to the schizont stage. Adding 90 mu L of culture medium and 10 mu L of inactivated and filter-sterilized immune serum into each hole of a 96-hole plate, preparing 8 holes for each sample, taking schizont period protozoa, adjusting the infection rate to be 2 percent, compacting RBC to 4 percent, adding 100 mu L into each hole after uniformly mixing, collecting RBC after culturing for 24 hours, staining a smear, and counting the infection rate of the annular bodies.
The invasion inhibition rate (normal culture well infection rate-experimental well infection rate/normal culture well infection rate) × 100%.
As shown in figure 2, the two monoclonal antibodies can obviously inhibit the invasion of plasmodium into erythrocytes, and the invasion inhibition rate is about 70%.
In conclusion, the monoclonal antibodies PfRH5-1 and PfRH5-2 obtained by the invention can obviously inhibit plasmodium from invading erythrocytes, reduce the infection rate of plasmodium, can detect plasmodium falciparum or screen corresponding medicines, and are the basis for clinically preventing or treating plasmodium falciparum related diseases.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A method for preparing a monoclonal antibody for inhibiting infection by plasmodium, comprising the steps of:
s1, acquisition of positive clones: synthesizing and cloning coding genes corresponding to the amino acid sequences of the sections 201 and 324aa of the PfRH5 protein sequence into a prokaryotic expression vector PGEX4T-1, selecting a positive cloning marker as PfRH5-1, synthesizing and cloning coding genes corresponding to the amino acid sequences of the sections 302 and 396aa of the PfRH5 protein sequence into the prokaryotic expression vector PGEX4T-1, and selecting a positive cloning marker as PfRH 5-2;
s2, prokaryotic expression:
respectively inoculating the two positive clone strains into an LB culture medium containing antibiotics for culture, adding an inducer IPTG (isopropyl-beta-thiogalactoside) for continuous culture overnight, crushing escherichia coli, centrifuging the crushed bacterial liquid, and respectively collecting supernatant and precipitate;
s3, purification of recombinant protein:
filtering the collected supernatant with a 0.22-micron filter membrane, passing through a column, collecting target proteins PfRH5-1-GST and PfRH5-2-GST, eluting, dialyzing, and concentrating to 1mg/mL to obtain the antigen;
s4, immunization of mice: continuously carrying out three times of immunization, wherein each time is 22 days, 10 days after the third time of immunization, detecting the tail tip of an immune mouse to take blood, selecting a mouse with higher antibody titer to carry out boosting immunization, wherein the dosage of boosting immunization antigen is 48-50 mu g/mouse, tail vein injection is carried out, and 180-200 mu l of blood is used;
s5, preparation of fused cells: taking a normal mouse, and preparing a spleen cell suspension for culturing;
s6, collecting the splenocytes from the immunized mice: obtaining immune mouse splenocytes, adding erythrocyte lysate into cell sediment for resuspension and mixing evenly, and counting after lysing erythrocytes;
s7, fusion culture and monoclonal screening;
s8, culturing the hybridoma to obtain the monoclonal antibodies PfRH5-1 and PfRH 5-2.
2. The method according to claim 1, wherein the culture conditions of both the positive clonal species in S2 are 37 ℃ and 250rpm, and the final concentration of the antibiotic in the LB medium is 100 g/mL.
3. The method according to claim 1, wherein in S3, the dialysis solution is PBS and the dialysis time is 12 h.
4. The method of claim 1, wherein the three immunizations in S4 are specifically: the first immune antigen amount is 100 mu g/mouse, and the subcutaneous injection is performed at multiple points, and the total amount is 200 mu l; the 2 nd to 3 rd immunization, the antigen amount is 100 mug/mouse, and the subcutaneous multi-point injection is performed for 200 mul.
5. The method according to claim 1, wherein the spleen cell suspension is cultured in S5 at an adjusted cell density of 1X 105Per ml, add to 96 well plate, 100. mu.l/well, 5% CO at 37 ℃2Culturing in an incubator.
6. The method according to claim 1, wherein in S7, the fusion culture and monoclonal screening are performed by: get 108Cells were prepared for fusion as described for SP2/0 cells: mixing splenocytes at a ratio of 1:10, centrifuging to obtain cell precipitate, preheating, sucking 1ml of PEG preheated to 37 deg.C, adding into the cell precipitate, mixing, standing in water bath at 37 deg.C for 60s, adding HAT culture medium to stop reaction, inoculating HAT culture medium containing fused cells into 96-well culture plate containing feeder layer cells, culturing at a concentration of 100 μ l/well and at 37 deg.C with 5% CO2Culturing in an incubator, performing monoclonal screening when the density of the culture medium occupies the holes 1/3, and selecting high-titer fusion positive holes.
7. The method according to claim 1, wherein in S8, the hybridoma cells are cultured by: selecting 10-week-old mice, inoculating incomplete adjuvant into abdominal cavity, wherein each mouse has an amount of 0.5ml, and injecting hybridoma cells diluted with serum-free culture medium into abdominal cavity, wherein the number of the injected cells is 2-3x106And collecting ascites after 7-10 days, centrifuging, collecting supernatant, and purifying to obtain monoclonal antibodies PfRH5-1 and PfRH 5-2.
8. The monoclonal antibody produced by the production method according to claim 1, wherein the monoclonal antibody comprises an antibody PfRH5-1 and an antibody PfRH 5-2.
9. Use of the monoclonal antibody of claim 8 in the preparation of a medicament for inhibiting or detecting infection by plasmodium falciparum.
10. Use of a monoclonal antibody according to claim 8 in the manufacture of a medicament for inhibiting plasmodium falciparum infection, wherein the infected subject is an erythrocyte.
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