Screening method and application of anti-plasmodium monoclonal antibody

Abstract

The invention discloses a screening method and application of an anti-plasmodium monoclonal antibody, and belongs to the technical field of genetic engineering and immunization. The screening method of the anti-plasmodium monoclonal antibody comprises the following steps: respectively designing upstream and downstream primers of each candidate antigen peptide fragment, and amplifying a target gene fragment through an enzyme chain polymerization reaction by taking a gDNA genome of plasmodium as a template; the method comprises the following steps: connecting BamH1 and Xho1 restriction enzyme cutting sites to a pET-28a vector with the same restriction enzyme cutting site, transforming and connecting plasmids through escherichia coli DH5a, identifying a recombinant substance through an enzyme chain polymerization reaction of upstream and downstream primers, and extracting successfully constructed plasmid DNA; transforming the successfully constructed antigen plasmid into a BL21 competent cell, and inducing antibody expression by using isopropyl-beta-D-thiogalactoside, so as to obtain a His fusion protein; and storing the purified protein. The anti-plasmodium monoclonal antibody provided by the invention has the characteristics of high affinity and strong specificity, provides a basis for screening and diagnosis of ovoid malaria, and has very high clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering and immunotechnology, and in particular to a screening method for anti-malaria parasite monoclonal antibodies and application thereof. Background Art

[0002] Plasmodium ovale is mainly distributed in tropical areas of countries such as West Africa and Southeast Asia. The malaria parasite is mainly divided into two subtypes: mutant Pocurtisi and traditional Powallikeri. The two subtypes have a common regional distribution, but the mutant Powallikeri can cause higher parasitemia and a higher single-species infection ratio. Mixed infections are often present. Since the incidence of ovale malaria in the population is lower than that of falciparum malaria and vivax malaria, it is often overlooked. However, there are more and more reports that ovale malaria infection can cause severe complications such as acute respiratory distress syndrome and lead to death.

[0003] Unlike falciparum malaria, ovale malaria is a type of malaria that will relapse even if it is completely cured. This is because ovale Plasmodium can persist in the patient's liver cells in a dormant state, and drugs such as artemisinin and chloroquine, which are the first-line drugs for treating falciparum malaria, cannot kill them. Only primaquine can kill dormant parasites. Under conditions such as low immunity of the patient, these dormant parasites will wake up, proliferate in the liver cells and be released into the blood, causing relapse and re-infection. Serological testing is a fast and effective method, but because most high serum reaction antigens such as MSP1-19 have a high degree of homology, although they can be used as good serological detection indicators, they cannot distinguish between infected Plasmodium species well, so they are not suitable for detecting mixed infected Plasmodium species. Therefore, accurate detection of ovale malaria and giving the correct malaria treatment plan will effectively prevent the recurrence of ovale malaria and achieve a radical cure.

[0004] In the current malaria prevention and control and monitoring work, malarial parasite microscopy is the gold standard for malaria diagnosis. It requires simple instruments and equipment, and has cheap consumables, making it more suitable for grassroots implementation. However, the traditional microscopy method has a low detection rate and is time-consuming. In addition, the professional quality and experience of the microscopy personnel will directly affect the determination of the results. When the parasite density is low, microscopy is prone to miss detection. Moreover, since the ovale malarial parasite is similar in morphology to several other malarial parasites, its infection type cannot be accurately identified by microscopy. Plasmodium nucleic acid detection uses polymerase chain reaction to detect specific target gene sequences through in vitro amplification. Its sensitivity and specificity are higher than those of microscopy, but most of them must be performed in the laboratory. Antigen detection is a simple, intuitive, and easy-to-master method with higher diagnostic accuracy and sensitivity than microscopy. Currently, most rapid diagnostic kits on the market use Plasmodium histidine-rich protein-2 (HRPII) and Plasmodium lactate dehydrogenase (LDH) as target antigens. Although they are intuitive and convenient, they can only diagnose Plasmodium vivax or Plasmodium falciparum, and cannot perform differential diagnosis on Plasmodium malariae and Plasmodium ovale. In addition, HRP-II has genetic polymorphisms that affect the test results.

[0005] Therefore, there is a need in the art for a detection reagent and related method for Plasmodium, especially Plasmodium ovale, with high specificity, high sensitivity and low cross-reaction, so as to achieve simple, rapid, sensitive and specific detection of Plasmodium ovale. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for screening anti-Plasmodium monoclonal antibodies and application thereof.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] A method for screening anti-malarial monoclonal antibodies comprises the following steps:

[0009] (1) Selection of target genes: Design upstream and downstream primers for each candidate antigen peptide segment, use gDNA of Plasmodium as template, and amplify the target gene fragment by enzyme chain reaction, with BamH1 and Xho1 as restriction sites;

[0010] (2) After the amplified product of the enzyme chain reaction is recovered, the BamH1 and Xho1 restriction sites are connected to the pET-28a vector with the same restriction sites, the connected plasmid is transformed by Escherichia coli DH5a, the recombinant plasmid is identified by enzyme chain reaction with the upstream and downstream primers, and the successfully constructed plasmid DNA is extracted;

[0011] (3) The successfully constructed antigen plasmid was transformed into BL21 competent cells, and the antibody expression was induced with isopropyl-β-D-thiogalactoside to obtain His fusion protein;

[0012] (4) The antigen peptide with his tag was purified using Ni-NTA agarose gel column to verify the purity of the purified protein.

[0013] The antigenic peptide segment includes any one of MSP1, MSP8, and GAMA; the MSP1 includes two subtypes of Poc and Pow, MSP8 includes two subtypes of Poc and Pow, and GAMA includes two subtypes of Poc and Pow.

[0014] The upstream and downstream primers include any one of MSP1-Poc-F and MSP1-Poc-R, MSP1-Pow-F and MSP1-Pow-R, MSP8-Poc-F and MSP8-Poc-R, MSP8-Pow-F and MSP8-Pow-R, GAMA-Poc-F and GAMA-Poc-R, GAMA-Pow-F and GAMA-Pow-R.

[0015] The nucleotide sequence of the MSP1-Poc-F is ATGAAGGTGTTCGTATTTGCG;

[0016] The nucleotide sequence of the MSP1-Poc-R is

[0017] TTAAAGTAAGTTAAATAGGATCAATAAGACTGC;

[0018] The nucleotide sequence of the MSP1-Pow-F is ATGAAGGTGTTCGTATTTGCG;

[0019] The nucleotide sequence of the MSP1-Pow-R is

[0020] TTAAAGTAAGTTAAATAGGATCAATAAGACTGC;

[0021] The nucleotide sequence of the MSP8-Poc-F is

[0022] ATGGTTATGATTATGAAAAAGAATTCGC;

[0023] The nucleotide sequence of the MSP8-Poc-R is

[0024] CTATAATAAATATAGACATAACATCACTATTATTAGAAAAATAAAACA;

[0025] The nucleotide sequence of MSP8-Pow-F is

[0026] ATGGTTATGATTATGAAAAAGAATTCGTATATAC;

[0027] The nucleotide sequence of the MSP8-Pow-R is

[0028] CTAGAATAAATATAGACATAATATCACTATTATTAGAAAAAATGAAA;

[0029] The nucleotide sequence of GAMA-Poc-F is

[0030] ATGAAATACAATACCGTTTTTACCGT;

[0031] The nucleotide sequence of GAMA-Poc-R is

[0032] CTATACGAGATAAAGGAAAATAGAAAAGAGAAC;

[0033] The nucleotide sequence of GAMA-Pow-F is

[0034] ATGAAATACAATACAGTTTTTACCGTGC;

[0035] The nucleotide sequence of GAMA-Pow-R is

[0036] CTATACGAGATAAAGGAAAATAGAAAAGAGAAAG.

[0037] The reaction conditions of the amplification are as follows: heating the reaction system to 98°C for 3 minutes, maintaining at 98°C for 10 seconds, annealing at 45°C for 1 minute, extending at 72°C for 1 minute, cycling 35 times, and finally reacting at 72°C for 5 minutes, cooling, and terminating the reaction.

[0038] An anti-Plasmodium monoclonal antibody is obtained according to the above-mentioned anti-Plasmodium monoclonal antibody screening method.

[0039] The recombinant amino acid sequence of the anti-malarial monoclonal antibody includes the amino acid sequence of the Poc subtype of MSP1 as shown in SEQ ID NO.1 in the sequence listing; the amino acid sequence of the Pow subtype of MSP1 as shown in SEQ ID NO.1SEQ ID NO.2 in the sequence listing; the amino acid sequence of the Poc subtype of MSP8 as shown in SEQ ID NO.3 in the sequence listing; the amino acid sequence of the Pow subtype of MSP8 as shown in SEQ ID NO.4 in the sequence listing; the amino acid sequence of the Poc subtype of GAMA as shown in SEQ ID NO.5 in the sequence listing; and the amino acid sequence of the Poc subtype of GAMA as shown in SEQ ID NO.6 in the sequence listing.

[0040] The recombinant nucleotide sequence encoding the anti-Plasmodium monoclonal antibody includes the nucleotide sequence of the Poc subtype of MSP1 such as SEQ ID NO.7 in the sequence listing; the nucleotide sequence of the Pow subtype of MSP1 such as SEQ ID NO.8 in the sequence listing; the nucleotide sequence of the Poc subtype of MSP8 such as SEQ ID NO.9 in the sequence listing; the nucleotide sequence of the Pow subtype of MSP8 such as SEQ ID NO.10 in the sequence listing; the nucleotide sequence of the Poc subtype of GAMA such as SEQ ID NO.11 in the sequence listing; and the nucleotide sequence of the Poc subtype of GAMA such as SEQ ID NO.12 in the sequence listing.

[0041] The invention relates to the use of the anti-malaria parasite monoclonal antibody in the preparation of a reagent for screening malaria, especially ovale malaria.

[0042] The beneficial effects of the present invention are: (1) compared with traditional anti-malarial monoclonal antibodies, the monoclonal antibodies prepared by the present invention can induce stronger humoral and cellular immune responses.

[0043] (2) By analyzing the antigen-antibody recognition characteristics of patients with ovale malaria, we can identify ovale-specific serological biomarkers and provide a solution for serological screening and diagnosis of ovale malaria and its subtypes.

[0044] (3) The anti-malarial monoclonal antibodies provided by the present invention can be mass-produced by biological methods for expression, purification and production, which greatly reduces the production cost. The preparation is simple, the antibody titer is high, the production cycle is short, and the antibody can be prepared on a large scale at a low price. Therefore, the antibody has great potential for clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0046] Figure 1 This is the SDS-PAGE image after cloning and purification of MSP1 as the antigen peptide;

[0047] Figure 2 This is the SDS-PAGE image after cloning and purification of MSP8 as an antigen peptide;

[0048] Figure 3 This is the SDS-PAGE image after cloning and purification of GAMA as the antigen peptide;

[0049] Figure 4This is a diagram showing the effect of inducing specific antibody IgG response at different serum dilution ratios;

[0050] Figure 5 The figure is a graph showing the affinity effects of different monoclonal antibodies injected;

[0051] Figure 6 The effect diagram of inducing specific antibody IgG response after injection of monoclonal antibody for different days of immunization;

[0052] Figure 7 This is a diagram showing the effect of inducing specific antibody IgG response after injection of different monoclonal antibodies;

[0053] Figure 8 This is a graph showing the effect of lymphocyte proliferation rate after injection of different monoclonal antibodies. DETAILED DESCRIPTION

[0054] Screening and identification of Plasmodium ovale biomarker antigen sequences:

[0055] (1) Based on the known high serum reaction markers of homologous malarial parasites, more than 200 proteins of Plasmodium ovale erythrocytic stage were selected and expressed. The amplified target gene fragments were connected to the pET-28a vector and expressed in BL21 Escherichia coli. The protein purity was purified by Ni-NTA agarose gel purification and verified by SDS-PAGE and Western blot experiments. The protein concentration was detected by BCA and stored at -80°C.

[0056] (2) Serum screening was carried out using protein chip technology, and the highly antigenic Plasmodium ovale proteins obtained through screening included: MSP1, GAMA, AMA-1, MSP8, GAMA, RALP1, and MTRAP;

[0057] (3) Use protein array to further identify the potential highly antigenic Plasmodium ovale molecules obtained in the initial screening, and through analysis with individual sera of Plasmodium ovale patients, identify molecules with the potential for serological molecular markers;

[0058] The amino acid sequences of some candidate proteins of Plasmodium ovale and other Plasmodium strains were compared and analyzed for polymorphism using BlastP software, and antigens with highly homologous sequences between species were excluded. The following is a homologous sequence comparison table:

[0059]

[0060]

[0061] In the experimental process, the present invention preferentially selects MSP1, MSP8 and GAMA with a homology of less than 80% as Plasmodium ovale proteins.

[0062] Example 1

[0063] Cloning, expression and purification of Plasmodium ovale specific recombinant antigen

[0064] Recombinant plasmid construction:

[0065] (1) Selection of target genes: Upstream and downstream primers of candidate antigenic peptides were designed and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The target gene fragments were amplified by enzyme chain reaction using gDNA of Plasmodium ovale Poc and Pow as templates, and BamH1 and Xho1 were used as restriction sites;

[0066] (2) After the amplified product of enzyme chain reaction was recovered, the BamH1 and Xho1 restriction sites were connected to the pET-28a vector with the same restriction sites, and the plasmid was transformed into Escherichia coli DH5a, and the recombinant plasmid was identified by enzyme chain reaction with the above and downstream primers. The monoclonal colony was picked and the positive clone was verified by restriction digestion and sequenced, and the successfully constructed plasmid DNA was extracted using a plasmid extraction kit (AXYGEN Company);

[0067] The Plasmodium ovale protein includes any one of MSP1, MSP8, and GAMA; the MSP1 includes two subtypes of Poc and Pow, MSP8 includes two subtypes of Poc and Pow, and GAMA includes two subtypes of Poc and Pow.

[0068] The upstream and downstream primers include any one of MSP1-Poc-F and MSP1-Poc-R, MSP1-Pow-F and MSP1-Pow-R, MSP8-Poc-F and MSP8-Poc-R, MSP8-Pow-F and MSP8-Pow-R, GAMA-Poc-F and GAMA-Poc-R, GAMA-Pow-F and GAMA-Pow-R.

[0069] The reaction conditions of the amplification are as follows: heating the reaction system to 98°C for 3 minutes, maintaining at 98°C for 10 seconds, annealing at 45°C for 1 minute, extending at 72°C for 1 minute, cycling 35 times, and finally reacting at 72°C for 5 minutes, cooling, and terminating the reaction.

[0070] The recombinant amino acid sequence thus obtained is as follows:

[0071] The recombinant amino acids obtained by cloning the Poc subtype of MSP1 are as follows:

[0072]

[0073] The recombinant amino acids obtained by cloning the Pow subtype of MSP1 are as follows:

[0074]

[0075] The recombinant amino acids obtained by cloning the Poc subtype of MSP8 are as follows:

[0076] MVMIMKKNSLILIFLIFSLIYYKNTVEGNIDIANGNGIININNNDSNGGNGSDKKIPPMNTGGNNDNMNKNNNVGGNDNGNNVDDLTNNSLKDDSKDNDENKNKKENNDIESKGDDNDNKE DNNSEKNDESLKKILNIVDEMENIQELLDGDSNILDKYNIKLVDEEDGDSKKKKLIGEYDLKMIKKVLLFREKISRTCENNFMNVNLTLKKCFNKDDPKLSKSCEKIKRGLSKNNMSIEDF ILGLLEDLFDKINDNFIQNDSFDLNDYLADFELINYLLLHESAELLKEIIHILDAINFKVESDALTKISNSAYSGMNINDKIKDDITNLLKMPSAKFFKIGIDKKTKMLIPVQAQHKGSSM KQFAYHFLDKNKVCEHTKCPLNSNCYVINSEETCRCLPGFSDVKIDNVMNCVRDDTMDCSNNNGGCDVNATCSLIDKKIVCECKENFEGDGIYCSNSILNSINCFIFLIIVMLCLYLL(SEQ ID NO.3);

[0077] The recombinant amino acids obtained by cloning the Pow subtype of MSP8 are as follows:

[0078] MVMIMKKNSYILIFLIFSLIYYKNTVEGNIDISNGNGIININNNDNNGGNGNDKNIPPMNTGGNNDNMNKNNNVGGNDNGNNVNDLTNNNLKDNSKDNDENKNKKENNDNESKGDDNDNKEDNNSEKNDESLKKILNIVDEMENIQELLDGNSNILDKYNIKLVDEEDGDSKKKKLIGEYDLKMIKKVLLFREKISRTCENNFMNVNVTLKKCFNKDDPKLSKSCEKIKRGLSKNNMSIEDFILGLLEDLFDKINDNFIQNDSFDLNDYLADFELINYLLLHESTELLKEIIHIIDAINFKVESDALTKISNSAYSGMNINDKIKDDITNLLKMPSAKFFKIGVDKKTKMLIPVQAQHKGSSMKQFAYHFLDKNKVCEHTKCPLNSNCYVIDAEETCRCLPGFSDVKIDNVMNCVRDDTIDCSNNNGGCDVNATCSLIDKKIVCECKENFEGDGIYCSNSILNSINCFIFLIIVILCLYLF(SEQ ID NO.4).

[0079] The recombinant amino acids obtained by protein cloning using the Poc subtype of GAMA are as follows:

[0080] MKYNTVFTVLLVIALFNAVNCLIRNPNSNSQALVPEQNNNQNGKPNGLSNNNDNTCEIQKMAEEMMEKLLNEKDVFTSIMEPLQSKLNDDYLCTQPKYTNICINEKDKTPLTFPCTSPKYENLIHEFTYKKLCHSKIAFSNTLLKSFIDQNKEENTFNNITQNYNTLLNCIDDDLKEIYKSSIDLFSDLKDSVVEITEKLWSKTITDVLKKREQLVTGIFCELRNGNKSSLVENSLSYENLGILKINNVQLLNEAYSAFSDYYYYFPIFATKLLEKGGLVERLVSIHENLTNYRTRNILKKINDQSKNEVLNNEELLNSLSNYKHHNQKNNGTFAQLKGIKISALEAASNGNNEAASGSAGGRANENINGSDNTSRSVNANENASQAQNEQQNQPQNQQQNHTQNQAQNQAQNQVQNQAQNQAQNQMQNHAQNQAQNQAQNQAQNQAQNQAQNEAQNEAQNEAQNQPQNQMQNQAQNQAQNQVQNQAQNEAQNQPQNEAHHRQQNEATNLSARVEQNNHISSSTNRVLNNPLYSTSSLKAKNVAQLVKDLLQSINIVKFENNEPTGQIDEEGIKKLIQNSFLDLSDNTMLVRLLIKPQAVILFIIQSFIMMTPSPTRDAKTFCRKALVNGQLIDSSDLDVASEEEDLINSFASKYNLIYEKIKMEELRENEQNKKALKTSKTHLSALEVRNTQNITQGISTENKLNSSRNAPLIAVVTDSASETTDDIIQNNVDLSALSPDVEQTFQTLNAKGGSFSANASYALLVLFSIFLYLV(SEQ ID NO.5);

[0081] The recombinant amino acids obtained by protein cloning with the Pow subtype of GAMA are as follows:

[0082] MKYNTVFTVLLVIALFSAVNCLIRNPNNNSQALVPEQNINQNGKPNGPSNNNDNTCEIQKMAEEMMEKLLNEKDVFTSIMEPLQSKLNDDYLCTQPKYTNICINEKDKTPLTFPCTSPKYENLIHEFTYKKLCHSKIAFSNTLLKSFIDQNKEENTFNNITQNYNTLLNCIDDDLKEIYKSSIDLFSDLKDSVVEITEKLWSKTITDVLKKREQLVTGIFCELRNGNKSSLVENSLSYENLGILKINNVQLLNEAYSAFSDYYYYFPIFATRLLEKGGLVERLVSIHENLTNYRTRNILKKINDQSKNEVLNNEELLNSLSNYKHHNQKNNGTFAQLKGIKISALEEASNGNNEAASGSAGGRANENINGSDSTSRSVNANANANASQAQNEQQKQPQNEQQKQPQNEQQKQQQSQQQSQQQSQQQSQQQNQQQSQEQNQAQNQAQNQAQNQVPNQSQNQAPNQSLNQSLNQSQNQSQNQSQNQSQNQSQNQSQNQAQNQSQNQAQNQAQNQAQNQAPNQSQNQMQNQAQNQVQNETQNEAHHHQQNEATNISAMAEHSNHISSSTNGALNNPLYSTSSLKAKNVAQLVKDLLQSINIVKFENNEPTGQIDEEGIKKLIQNSFLDLSDNTMLVRLLIKPQAVILFIIQSFIMMTPSPTRDAKTFCKKALVNGQLIDSSDLDVASEEEDLINSFASKYNLIYEKIKMEELRENEQNKKALKTSKTHLSALEVRNTQNITQGMSTENKLNSSRNAPLIAVVTDSASETTDDIIQNNVDLSALSPDVEQTFQTLNTKNGSLSANASYALLFLFSIFLYLV(SEQ ID NO.6).

[0083] The recombinant coding nucleotide sequence obtained by cloning the Poc subtype of MSP1 is as follows:

[0084]

[0085] The recombinant coding nucleotide sequence obtained by cloning the Pow subtype of MSP1 as protein is as follows:

[0086]

[0087] The recombinant coding nucleotide sequence obtained by cloning the Poc subtype of MSP8 as a protein is as follows:

[0088]

[0089] The recombinant coding nucleotide sequence obtained by cloning the Pow subtype of MSP8 as protein is as follows:

[0090]

[0091] The recombinant coding nucleotide sequence obtained by cloning the Poc subtype of GAMA as a protein is as follows:

[0092]

[0093] The recombinant coding nucleotide sequence obtained by cloning the Pow subtype of GAMA as protein is as follows:

[0094]

[0095] Expression and purification of recombinant proteins:

[0096] (3) The successfully constructed antigen plasmid was transformed into BL21 competent cells, and antibody expression was induced with isopropyl-β-D-thiogalactoside. The supernatant (periplasmic extract) was collected, and the periplasmic extract was dialyzed into PBS and purified using His tag agarose gel to obtain His fusion protein. SDS-PAGE and Western blot experiments verified that the target protein was successfully expressed. The results are shown in Figure 1 , Figure 2 and Figure 3 ;

[0097] (4) The antigen peptide with his tag was purified by Ni-NTA agarose gel (QIAGEN), the purity of the purified protein was verified by SDS-PAGE, the protein concentration was detected by BCA, and the protein was stored at -80°C.

[0098] Example 2: Binding ability of antibodies to TRAg proteins: MSP1 was used preferentially during the experiment. 19 Antigen protein was used as the whole experiment.

[0099] (1) Coating 96-well ELISA plate (TRAg protein as antigen):

[0100] ①100ng antigen / well, 200ul / well;

[0101] ②It needs to be placed at 4℃ overnight to complete the coating;

[0102] ③ Two groups need to be set up, the urea-treated group and the urea-untreated group. The urea-untreated group can be used as the last IgG content test;

[0103] ④ The serum dilution ratio is selected according to the antibody titer value;

[0104] (2) Blocking: Take out the 96-well ELISA plate from the 4°C refrigerator, wash each well with a rinse solution of 0.1% Tween-20 and 1×TBS, and after tapping the plate for the last time, add 200 ul / well of blocking solution, which is composed of 5% skimmed milk powder and 1×TBS;

[0105] (3) Serum incubation (antibody incubation): Wash each reaction well three times with a rinse solution of 0.1% Tween-20 and 1×TBS, dilute the serum stored at -80°C to the optimal dilution factor of 1:10,000, add 100 μl / well of the diluted serum to the antigen-coated wells, and incubate at room temperature for 2 h. The specific antibody binds to the antigen to form a solid-phase antigen-antibody complex;

[0106] (4) Urea elution: After the primary antibody incubation, wash twice and then add urea. The urea-treated plate is added with 6 mol / L urea aqueous solution at 100 ul / well and incubated for 10 min. The untreated plate is washed three times with 200 ul / well using TBST.

[0107] (5) Adding enzyme-labeled secondary antibody: Wash each reaction well three times with TBST rinse solution at 200 ul / well. After washing, only specific antibodies remain on the solid phase carrier. Other immunoglobulins and impurities in the serum are washed away during the washing process because they cannot bind to the solid phase antigen. Add 100 ul / well of horseradish peroxidase-labeled goat anti-mouse IgG (H+L) secondary antibody with a dilution ratio of 1:5000 and incubate at room temperature for 1.5 h.

[0108] (6) Color development with substrate solution: Wash each reaction well three times with 200 μl / well of TBST rinse solution. The amount of enzyme on the solid phase carrier after washing represents the amount of specific antibody. Add 100 μl / well of TMB substrate color development solution to each reaction well and develop the color for 2 min in the dark.

[0109] (7) Termination of reaction: After the reaction well changes color, add 50ul / well of 2mol / L sulfuric acid stop solution to terminate the reaction;

[0110] (8) OD determination by microplate reader 450nm : The results can be observed directly with the naked eye on a white background.

[0111] from Figure 4 It can be seen that the OD after serum gradient dilution 450nm As the dilution ratio increases, OD 450nm The value is getting smaller and smaller, that is, the binding force of the specific antibody is getting weaker, the antibody concentration is getting smaller and smaller, the dilution concentration is getting higher, and the immune response level is getting lower and lower.

[0112] from Figure 5 It can be seen that the antibodies produced by the test group mice are sensitive to rPocMSP1 19 -GST has extremely high affinity, with an affinity value close to 100%, showing excellent affinity. The antibodies produced in mice have strong binding ability. The immunization strategy used (i.e., the combination of key proteins of Plasmodium ovale and Freund's adjuvant) can effectively stimulate the immune response of mice and produce antibodies with extremely high affinity, indicating that the screened monoclonal antibodies can specifically bind to TRag protein.

[0113] Example 3: Serological response to Plasmodium ovale-specific antigens and detection of antibody levels

[0114] (1) Collect serum from hospital-acquired patients infected with malarial parasites or collect filter paper blood from patients infected with malarial parasites. Cut the filter paper soaked with blood into small pieces, soak them in 400ul PBST buffer for 12 hours, centrifuge at 800rpm, and then draw the supernatant as the filter paper blood supernatant to be tested. At the same time, collect serum from normal subjects who are not infected with malarial parasites by venous blood sampling;

[0115] (2) Protein-array detection of the serum cross-reactivity level of candidate antigens among Plasmodium species: The experiment used normal human serum reactions as the negative control group, and serum reactions infected with Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae as the positive experimental group. The purified antigen peptide protein was diluted with 0.05 mol / L Na2CO3 buffer at pH 9.6 to a concentration of 1 ug / mL, and 100 ul / well was added to a 96-well ELISA plate. The plate was coated overnight at 4°C, blocked with 200 ul / well blocking solution at room temperature for 2 h, washed three times with 0.1% TBST buffer, and diluted with 1:100. Serum or filter paper blood supernatant was diluted in a ratio of 0.00, and added to the corresponding wells at 100ul / well, and incubated at room temperature for 2h; after washing 3 times with 0.1% TBST buffer, 100ul / well was added with horseradish peroxidase-labeled goat anti-mouse IgG (H+L) secondary antibody with a dilution ratio of 1:5000, and incubated at room temperature for 90min, after washing 3 times with 0.1% TBST buffer, 100ul / well was added with TMB substrate to develop color at room temperature in the dark for 2min, 50ul / well was added with 2mol / L sulfuric acid stop solution to terminate the reaction, and the OD of all wells was detected at a wavelength of 450nm using an enzyme reader. 450nm value;

[0116] (3) Detection of Plasmodium ovale-specific antibody levels: The experiment used normal human serum reactions as the negative control group and Plasmodium ovale-infected serum reactions as the positive experimental group. The purified antigen peptide protein was diluted with 0.05 mol / L Na2CO3 buffer at pH 9.6 to a concentration of 1 ug / mL. 100 ul / well was added to a 96-well ELISA plate and coated overnight at 4°C. The plate was blocked with 200 ul / well blocking solution at room temperature for 2 h. The plate was washed three times with 0.1% TBST buffer and the serum or filter paper blood supernatant was diluted at a ratio of 1:10000. 100ul / well was added to the corresponding wells and incubated at room temperature for 2h; after washing 3 times with 0.1% TBST buffer, 100ul / well was added with horseradish peroxidase-labeled goat anti-mouse IgG (H+L) secondary antibody with a dilution ratio of 1:5000 and incubated at room temperature for 90min; after washing 3 times with 0.1% TBST buffer, 100μl / well TMB substrate was used for color development at room temperature in the dark for 2min; 50μl / well was added with 2mol / L sulfuric acid stop solution to terminate the reaction, and the OD of all wells was detected at a wavelength of 450nm using an enzyme reader. 450nm value;

[0117] (4) Analyze the level of serum cross-reactivity between Plasmodium species and the level of Plasmodium ovale-specific antibodies to screen out Plasmodium ovale-specific serum reaction markers with low intermediate cross-reactivity and high antibody production levels.

[0118] Results Figure 6 As shown, the mouse sera at 20 and 50 days after immunization are shown to be sensitive to several different antigens (rPocMSP1 19 -GST, rPowMSP1 19 -GST, GST, PBS) reaction, from the results we can see that rPowMSP1 19 -GST showed almost no change in immune response level 30 days after immunization, while rPocMSP1 19 -GST immune response level increased from 20 days to about 50 days, indicating that rPocMSP1 19 -GST as a recombinant protein produces significantly higher antibody levels in mice than rPowMSP1 19 -GST has a high antibody level and is more capable of inducing the production of antibodies, resulting in a better immune response level.

[0119] The results are as follows Figure 7 The results show that the immune response induced by the two recombinant proteins under the same conditions was compared. It can be seen from the results that the recombinant protein rPocMSP1 19 -GST produces significantly higher levels of antibodies in mice than rPowMSP1 19 -GST has high antibody levels and induces specific humoral immune responses.

[0120] Example 4: Detection of immunogenicity levels of key molecules of Plasmodium ovale

[0121] (1) Male BALB / C mice aged 6-8 weeks and weighing about 16 g were divided into an experimental group and a control group, with 6 mice in each group. The purified Plasmodium ovale recombinant protein was concentrated to 1 mg / mL and mixed with an equal volume of Freund's adjuvant. Each mouse was injected subcutaneously at multiple points and intraperitoneally with 50 μg. The first immunization was performed at week 0, and booster immunization was performed at week 3 and week 6.

[0122] (2) Detection of mouse-specific humoral immune response: At 1, 4, and 7 weeks after immunization, the mice were tail-clipped and blood was collected from the submandibular area of ​​the mice using a 5-ml syringe needle. The blood samples were incubated at 37°C for 30 min and then centrifuged at 3000 g for 30 min. The mouse serum was collected in a clean EP tube and stored at -80°C. The specific IgG level was detected using a conventional enzyme-linked immunosorbent assay, in which 50 ng / mL oval plasmodium key protein was coated and the mouse serum was incubated at a dilution of 1:10,000. The end point titration method was used to detect the production of specific antibodies.

[0123] The results are as follows Figure 8 As shown in the figure, ConA is used as a positive control. ConA can usually strongly activate T cells and B cells, so its lymphocyte proliferation rate may be higher; GST is used as a negative control. The lymphocyte proliferation rate of GST should be lower because it is not immunogenic. 19 -GST and rPowMSP1 19 -GST, the two recombinant proteins, had relatively high lymphocyte proliferation rates, which were much higher than those of the ConA positive control group and the GST negative control group, indicating that they induced an immune response in mice and could be used to evaluate their immunogenicity and ability to activate immune cells. The experimental group was able to induce the production of higher antibodies and specific humoral immune responses, providing a basis for serological biomarkers of Plasmodium ovale.

Claims

1. A method for screening anti-malarial monoclonal antibodies, characterized in that: The steps include: (1) Selection of target genes: Design upstream and downstream primers for each candidate antigen peptide segment, use gDNA of Plasmodium as template, and amplify the target gene fragment by enzyme chain reaction, with BamH1 and Xho1 as restriction sites; (2) After the amplified product of the enzyme chain reaction is recovered, the BamH1 and Xho1 restriction sites are connected to the pET-28a vector with the same restriction sites, the connected plasmid is transformed by Escherichia coli DH5a, the recombinant plasmid is identified by enzyme chain reaction with the upstream and downstream primers, and the successfully constructed plasmid DNA is extracted; (3) The successfully constructed antigen plasmid was transformed into BL21 competent cells, and the antibody expression was induced by isopropyl-β-D-thiogalactoside to obtain His fusion protein; (4) The antigen peptide with his tag was purified using Ni-NTA agarose gel column to verify the purity of the purified protein.

2. The method for screening anti-Plasmodium monoclonal antibodies according to claim 1, characterized in that: The antigenic peptide segment includes any one of MSP1, MSP8, and GAMA; the MSP1 includes two subtypes of Poc and Pow, MSP8 includes two subtypes of Poc and Pow, and GAMA includes two subtypes of Poc and Pow.

3. The method for screening anti-malarial monoclonal antibodies according to claim 1, characterized in that: The upstream and downstream primers include any one of MSP1-Poc-F and MSP1-Poc-R, MSP1-Pow-F and MSP1-Pow-R, MSP8-Poc-F and MSP8-Poc-R, MSP8-Pow-F and MSP8-Pow-R, GAMA-Poc-F and GAMA-Poc-R, GAMA-Pow-F and GAMA-Pow-R.

4. The method for screening anti-malarial monoclonal antibodies according to claim 1, characterized in that: The nucleotide sequence of the MSP1-Poc-F is ATGAAGGTGTTCGTATTTGCG; The nucleotide sequence of the MSP1-Poc-R is TTAAAGTAAGTTAAATAGGATCAATAAGACTGC; The nucleotide sequence of the MSP1-Pow-F is ATGAAGGTGTTCGTATTTGCG; The nucleotide sequence of the MSP1-Pow-R is TTAAAGTAAGTTAAATAGGATCAATAAGACTGC; The nucleotide sequence of the MSP8-Poc-F is ATGGTTATGATTATGAAAAAGAATTCGC; the nucleotide sequence of the MSP8-Poc-R is CTATAATAAATATAGACATAACATCACTATTATTAGAAAAATAAAACA; the nucleotide sequence of the MSP8-Pow-F is ATGGTTATGATTATGAAAAAGAATTCGTATATAC; The nucleotide sequence of the MSP8-Pow-R is CTAGAATAAATATAGACATAATATCACTATTATTAGAAAAATGAAA; The nucleotide sequence of the GAMA-Poc-F is ATGAAATACAATACCGTTTTTACCGT; the nucleotide sequence of the GAMA-Poc-R is CTATACGAGATAAAGGAAAATAGAAAAGAGAAC; The nucleotide sequence of the GAMA-Pow-F is ATGAAATACAATACAGTTTTTACCGTGC; the nucleotide sequence of the GAMA-Pow-R is CTATACGAGATAAAGGAAAATAGAAAAGAGAAAG.

5. The method for screening anti-malarial monoclonal antibodies according to claim 1, characterized in that: The reaction conditions of the amplification are as follows: heating the reaction system to 98°C for 3 minutes, maintaining at 98°C for 10 seconds, annealing at 45°C for 1 minute, extending at 72°C for 1 minute, cycling 35 times, and finally reacting at 72°C for 5 minutes, cooling, and terminating the reaction.

6. An anti-malarial monoclonal antibody, characterized in that: Obtained according to the screening method for anti-Plasmodium monoclonal antibodies according to any one of claims 1 to 5.

7. The anti-malarial monoclonal antibody according to claim 4, characterized in that: The recombinant amino acid sequence of the anti-malarial monoclonal antibody includes the amino acid sequence of the Poc subtype of MSP1 as shown in SEQ ID NO.1 in the sequence listing; the amino acid sequence of the Pow subtype of MSP1 as shown in SEQ ID NO.1SEQ ID NO.2 in the sequence listing; the amino acid sequence of the Poc subtype of MSP8 as shown in SEQ ID NO.3 in the sequence listing; the amino acid sequence of the Pow subtype of MSP8 as shown in SEQ ID NO.4 in the sequence listing; the amino acid sequence of the Poc subtype of GAMA as shown in SEQ ID NO.5 in the sequence listing; and the amino acid sequence of the Poc subtype of GAMA as shown in SEQ ID NO.6 in the sequence listing.

8. The anti-malarial monoclonal antibody according to claim 6, characterized in that The recombinant nucleotide sequence encoding the anti-Plasmodium monoclonal antibody includes the nucleotide sequence of the Poc subtype of MSP1 such as SEQ ID NO.7 in the sequence listing; the nucleotide sequence of the Pow subtype of MSP1 such as SEQ ID NO.8 in the sequence listing; the nucleotide sequence of the Poc subtype of MSP8 such as SEQ ID NO.9 in the sequence listing; the nucleotide sequence of the Pow subtype of MSP8 such as SEQ ID NO.10 in the sequence listing; the nucleotide sequence of the Poc subtype of GAMA such as SEQ ID NO.11 in the sequence listing; and the nucleotide sequence of the Poc subtype of GAMA such as SEQ ID NO.12 in the sequence listing.

9. Use of the anti-Plasmodium monoclonal antibody according to any one of claims 6 to 8 in the preparation of a reagent for screening malaria, especially Plasmodium ovale.

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