A Plasmodium falciparum RIFIN polypeptide 8-RIFIN and its derivative products and applications

Abstract

The present invention provides a Plasmodium falciparum (P.falciparum) RIFIN polypeptide 8-RIFIN and its derivative products and applications, belonging to the field of biological protein engineering technology. The Plasmodium falciparum RIFIN polypeptide 8-RIFIN provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 1, and is derived from the RIFIN peptide segment located at 243-267aa encoded by the RIFIN gene PF3D7_0900200 of Plasmodium falciparum 3D7. The polypeptide 8-RIFIN not only has a good ability to specifically identify the whole blood of patients infected with falciparum malaria, but also can bind to the immunosuppressive receptor LILRB1, which indicates that the polypeptide of the present invention is the key region for the binding of Plasmodium falciparum RIFIN protein to LILRB1, laying a foundation for further studying the molecular mechanism of Plasmodium falciparum using RIFIN protein for immune escape.

Description

Technical Field

[0001] The invention belongs to the technical field of biological protein engineering, and specifically relates to a Plasmodium falciparum RIFIN polypeptide 8-RIFIN and derivative products and applications thereof. Background Art

[0002] Protein-protein interaction (PPI) refers to the process in which two or more protein molecules form a protein complex through non-covalent bonds. Studying protein interactions can better reveal the functions of proteins and decode life activities. Only when the interactions between proteins proceed smoothly can the normal life activities of cells be guaranteed.

[0003] Plasmodium falciparum has become the most dangerous malarial parasite due to its high mortality rate and widespread resistance to antimalarial drugs, and is dominant in Africa, the continent with the most severe malaria in the world. The main reason for the morbidity and even mortality caused by Plasmodium falciparum infection is that the exponential growth of the malarial parasite increases the number of infected erythrocytes, leading to severe anemia, the adhesion of mature Plasmodium falciparum infected erythrocytes to microvascular endothelial cells, leading to blockage of the microvenous vascular system, and the formation of rosettes between infected and uninfected erythrocytes, resulting in blood flow obstruction. In its pathogenic mechanism, RIFIN proteins of the variant surface antigen family (P. falciparum-encoded repetitive interspersed families of polypeptides) play an important role. They can mediate the adhesion and rosette formation of infected erythrocytes in the microvenous vascular system, thereby blocking blood flow and occluding blood vessels to cause severe malaria. RIFIN protein is expressed on the surface of infected red blood cells and is one of the important immune targets. However, due to the polygenicity and gene polymorphism of the repetitive interspersed gene family rif (repetitive interspersed family), RIFIN protein can escape the attack of the immune system. At the same time, the binding of RIFIN protein to inhibitory receptors on the surface of immune cells, such as Leukocyte immunoglobulin-like receptor B1 (LILRB1) and Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), can downregulate the immune response and achieve the purpose of immune escape.

[0004] In recent years, with the continuous emergence of drug-resistant Plasmodium falciparum, the research and development of new treatment options has become the key to achieving malaria control and elimination plans. RIFIN proteins of the variant surface antigen family involved in the immune evasion mechanism of Plasmodium falciparum may also become important candidates for biological treatment and malaria vaccine proteins. At present, there are no reports on the key regions where the Plasmodium falciparum RIFIN protein binds to the immunosuppressive receptor LILRB1, which hinders the exploration of the molecular mechanism by which Plasmodium falciparum uses the variant surface antigen family RIFIN proteins for immune evasion. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a Plasmodium falciparum RIFIN polypeptide 8-RIFIN, which can specifically bind to the immunosuppressive receptor LILRB1, to provide a basis for studying Plasmodium falciparum immune escape and biological treatment.

[0006] The present invention provides a Plasmodium falciparum RIFIN polypeptide 8-RIFIN, and the amino acid sequence of the 8-RIFIN is shown in SEQ ID NO:1.

[0007] The present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in preparing a kit for detecting Plasmodium falciparum.

[0008] Preferably, the detection sample of Plasmodium falciparum is whole blood or serum of human or animal.

[0009] The invention provides a Plasmodium falciparum detection kit based on immunoassay technology, comprising the Plasmodium falciparum RIFIN polypeptide 8-RIFIN.

[0010] Preferably, the immunoassay technique comprises one or more of the following methods: enzyme-linked immunosorbent assay, immunoblotting, immunoenzyme staining, indirect fluorescent antibody detection and immunodiffusion.

[0011] Preferably, when the detection is based on an enzyme-linked immunosorbent assay, the kit comprises a detection plate coated with the Plasmodium falciparum RIFIN polypeptide 8-RIFIN, a washing solution, an enzyme-labeled secondary antibody and a display solution.

[0012] The present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in the preparation of a vaccine for preventing and treating Plasmodium falciparum.

[0013] The invention provides a vaccine for preventing and treating Plasmodium falciparum. The vaccine comprises the Plasmodium falciparum RIFIN polypeptide 8-RIFIN and an adjuvant.

[0014] The present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in preparing a reagent that specifically binds to the immunosuppressive receptor LILRB1.

[0015] The present invention provides the use of the polypeptide specifically binding to the immunosuppressive receptor LILRB1 in the application in preparing a reagent or a kit for blocking the immune escape of Plasmodium falciparum.

[0016] The Plasmodium falciparum RIFIN polypeptide 8-RIFIN provided by the present invention is a RIFIN peptide segment located at 243-267aa encoded by the Plasmodium falciparum RIFIN gene PF3D7_0900200, and the amino acid sequence is shown in SEQ ID NO: 1, and the amino acid sequence length is 25 amino acids. Experiments show that compared with the control mutant polypeptide (9-RIFIN), the polypeptide of the present invention not only has a better ability to specifically recognize the whole blood of patients infected with falciparum malaria, but also can bind to the immunosuppressive receptor LILRB1. This shows that the polypeptide 8-RIFIN provided by the present invention is the key region for the binding of the Plasmodium falciparum RIFIN protein to LILRB1, laying a foundation for further studying the molecular mechanism of Plasmodium falciparum using the RIFIN protein for immune escape. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The molecular weight results of mass spectrometry analysis of polypeptide fragments 8-RIFIN and 9-RIFIN are shown;

[0018] Figure 2 The results of antibody reactions to polypeptide fragments 8-RIFIN and 9-RIFIN;

[0019] Figure 3 It is the binding reaction diagram of polypeptide fragments 8-RIFIN and 9-RIFIN with inhibitory receptor LILRB1;

[0020] Figure 4 This is the binding reaction diagram of the polypeptide fragment 8-RIFIN and the polyclonal antibody of the immunized rabbit serum. DETAILED DESCRIPTION

[0021] The invention provides a Plasmodium falciparum RIFIN polypeptide 8-RIFIN, and the amino acid sequence of the 8-RIFIN is shown in SEQ ID NO: 1 (YQGSQCGSLSMLLGKSKPFCTFVEG).

[0022] In the present invention, the polypeptide 8-RIFIN is from Plasmodium falciparum 3D7, and the Plasmodium falciparum RIFIN peptide segment located at 243-267aa encoded by the RIFIN gene PF3D7_0900200 has an amino acid sequence length of 25aa. Five amino acid mutation sites (K259N, F261Y, F264L, E266N and G267E) are carried out based on the Plasmodium falciparum RIFIN peptide segment located at 243-267aa encoded by the RIFIN gene PF3D7_0900200 to obtain a mutant Plasmodium falciparum RIFIN polypeptide (9-RIFIN), and the amino acid sequence is shown in SEQ ID NO: 2 (YQGSQCGSLSMLLGKSNPYCTLVNE). Experiments on the binding reaction of polypeptides 8-RIFIN and 9-RIFIN with the LILRB1 receptor were carried out respectively, and the results showed that the polypeptide 8-RIFIN can specifically bind to the LILRB1 receptor, while the mutant polypeptide (9-RIFIN) cannot bind to the LILRB1 receptor, indicating that the polypeptide 8-RIFIN provided by the present invention is the key region for the binding of RIFIN protein to the LILRB1 receptor, and can serve as the basis for further studying the molecular mechanism of Plasmodium falciparum using RIFIN protein for immune evasion, and has important application value in the elimination and control of malaria.

[0023] In the present invention, the wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN and the mutant Plasmodium falciparum RIFIN polypeptide 9-RIFIN react with antibodies of whole blood of patients with falciparum malaria and normal human serum, respectively. The antibody reactions of polypeptides 8-RIFIN and 9-RIFIN with whole blood of patients with falciparum malaria show obvious differences. The antibody reaction positive rate of polypeptide 8-RIFIN with whole blood of patients with falciparum malaria is significantly higher than that of polypeptide 9-RIFIN. Therefore, the present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in the preparation of a kit for detecting Plasmodium falciparum. The detection sample of Plasmodium falciparum is preferably whole blood or serum of human or animal.

[0024] In the present invention, the wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN is subjected to immunogenicity experiments, the polypeptide 8-RIFIN is used as an immune antigen to immunize New Zealand white rabbits to prepare rabbit polyclonal antibodies, and the wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN is used to react with the immune rabbit polyclonal antibodies, and the results show that 8-RIFIN has sufficiently strong immunogenicity to New Zealand rabbits. Therefore, the present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in the preparation of a vaccine for the prevention and treatment of Plasmodium falciparum.

[0025] The invention provides a Plasmodium falciparum detection kit based on immunoassay technology, comprising the Plasmodium falciparum RIFIN polypeptide 8-RIFIN.

[0026] In the present invention, the detection mechanism of the detection kit is to use the specific binding of polypeptide 8-RIFIN with Plasmodium falciparum antibodies in the sample to be tested to achieve the purpose of detecting whether the sample is infected with Plasmodium falciparum. The present invention has no special restrictions on the specific types of immunoassay techniques, and can use immunoassay techniques known in the art, such as enzyme-linked immunosorbent assay, immunoblotting, immunoenzyme staining, indirect fluorescent antibody detection, and immunodiffusion. In the embodiment of the present invention, the specific effect of using polypeptide 8-RIFIN to detect samples infected with Plasmodium falciparum is described by taking enzyme-linked immunosorbent assay as an example. When the detection is based on the enzyme-linked immunosorbent assay, the kit preferably includes a detection plate coated with the Plasmodium falciparum RIFIN polypeptide 8-RIFIN, a washing solution, an enzyme-labeled secondary antibody, and a display solution. The present invention has no special restrictions on the preparation method of the kit, and can use the indirect detection method known in the art to prepare the kit. The coating concentration of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN is preferably 1 to 5 μg / ml, more preferably 2 to 3 μg / ml. During coating, the solvent of the polypeptide 8-RIFIN is a coating buffer. The coating buffer is preferably 0.05M carbonate buffer (pH 9.6). The coating is preferably coated at 4°C for 10 to 14 hours. After the coating, the test plate is preferably blocked. The blocking solution is preferably a PBS solution containing 1% BSA by mass concentration. The blocking condition is incubated at 37°C for 2 hours. The present invention has no special restrictions on the use of the kit, and the indirect enzyme-linked immunosorbent assay kit known in the art can be used. The kit was used for detection, and the results showed that the positive rate of the immune reaction of the wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN with the whole blood of patients with falciparum malaria was 62.5% (75 / 120), and the false positive rate of the immune reaction with normal human serum was 10.9% (5 / 46), and there was a significant difference between the two (P<0.0001). The positive rate and false positive rate of the mutant Plasmodium falciparum RIFIN polypeptide 9-RIFIN with the above two groups of samples were 29.2% (35 / 120) and 4.3% (2 / 46), respectively, and there was a significant difference between the two (P = 0.0006). The above results show that the IgG antibody reaction of 8-RIFIN and 9-RIFIN in the whole blood of patients with falciparum malaria and the serum of normal people is significantly different.

[0027] The present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in the preparation of a vaccine for preventing and treating Plasmodium falciparum.

[0028] The present invention provides a vaccine for preventing and treating Plasmodium falciparum, the vaccine comprising the Plasmodium falciparum RIFIN polypeptide 8-RIFIN and an adjuvant. A polypeptide 8-RIFIN fragment is selected for immunization of New Zealand rabbits, two New Zealand rabbits are immunized in total, and each rabbit is immunized four times to obtain corresponding immune rabbit serum polyclonal antibodies R1 and R2, and this process is completed by Shanghai Youke Biotechnology Co., Ltd. The obtained rabbit polyclonal antibodies R1 and R2 are subjected to antibody reaction tests with wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN, and the results show that 8-RIFIN has sufficiently strong immunogenicity to New Zealand rabbits.

[0029] The present invention has no particular limitation on the method for preparing the vaccine, and any method for preparing a polypeptide vaccine known in the art may be used.

[0030] The present invention provides the use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN in preparing a reagent that specifically binds to the immunosuppressive receptor LILRB1.

[0031] In the present invention, the recombinantly expressed immunosuppressive receptor LILRB1 is used as the detection object, and the binding of polypeptide 8-RIFIN to receptor LILRB1 is detected based on the enzyme-linked immunosorbent assay method. The results show that polypeptide 8-RIFIN can significantly bind to LILRB1 receptor, while mutant polypeptide 9-RIFIN hardly binds to receptor LILRB1. It can be seen that polypeptide 8-RIFIN is the key region for the binding of Plasmodium falciparum RIFIN protein to LILRB1, and the study of the molecular mechanism of Plasmodium falciparum using RIFIN protein for immune evasion has important application value in malaria elimination and control.

[0032] The present invention provides the use of a polypeptide specifically binding to the immunosuppressive receptor LILRB1 in the application in the preparation of a reagent or kit for blocking the immune escape of Plasmodium falciparum. After binding to the immunosuppressive receptor LILRB1, the polypeptide 8-RIFIN blocks the binding of Plasmodium falciparum RIFIN protein to the receptor LILRB1, exposing the Plasmodium falciparum RIFIN protein and preventing it from escaping immune cell recognition, thereby increasing the probability of immune response.

[0033] The present invention has no special restrictions on the preparation of the reagent or kit, and the reagent or kit for blocking immune escape known in the art can be used.

[0034] The Plasmodium falciparum RIFIN polypeptide 8-RIFIN and its derivative products and applications provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Antibody Immune Response to Plasmodium falciparum RIFIN Peptide 8-RIFIN

[0037] 1 Materials

[0038] 1.1 Plasmodium falciparum RIFIN protein peptides 8-RIFIN and 9-RIFIN were synthesized by GenScript Biotech Co., Ltd. (Nanjing).

[0039] 1.2 ELISA plate (96 wells) and sealing film were purchased from Thermo SCIENTIFIC.

[0040] 1.3 Blood samples from patients with Plasmodium falciparum and normal subjects were collected from malaria-endemic areas in Togo.

[0041] 1.4 Main reagents and enzyme tools

[0042] Reagent name Reagent Source 20×PBS Buffer Sangon Biotech Tween-20 Sangon Biotech Soluble one-component TMB substrate solution TIANGEN Goat anti-human IgG H&L (HRP conjugated) Abcam BSA Solarbio Dimethyl sulfoxide,Analytical grade SIGMA <![CDATA[Na2CO3]]> Shanghai Jingxi Chemical <![CDATA[NaHCO3]]> Shanghai Jingxi Chemical <![CDATA[H2SO4]]> Sinopharm Other reagents All are domestic analytical reagents

[0043] 2 Methods

[0044] 2.1 Preparation of Plasmodium falciparum RIFIN polypeptide 8-RIFIN and 9-RIFIN working solution

[0045] 1) Preparation of peptide master solutions: Place 8-RIFIN and 9-RIFIN peptide tubes containing 1 mg of powder in a 4°C centrifuge and centrifuge at 12,000 rpm for 15 min. Then, according to the recommendations in the peptide synthesis report, 8-RIFIN is dissolved in 500 μl of ultrapure water to a concentration of 2 mg / ml, and 9-RIFIN is dissolved in 500 μl of dimethyl sulfoxide to a concentration of 2 g / ml.

[0046] 2) Preparation of peptide working solution: dilute 8-RIFIN and 9-RIFIN stock solutions with coating buffer prepared in advance and placed in a 4°C refrigerator, dilute the peptide stock solution 1000 times with coating buffer, and prepare 2 μg / ml peptide working solutions.

[0047] 2.2 Antibody immune response to Plasmodium falciparum RIFIN polypeptides 8-RIFIN and 9-RIFIN

[0048] 1) Coating: Mark the peptide type on the side of the 96-well plate, add the prepared 2 μg / ml peptide working solution to the corresponding wells of the 96-well plate at a volume of 100 μl / well, of which the first two wells in each plate are 100 μl of coating solution as blank control, and seal the plate with a sealing film and place it steadily in a 4°C refrigerator overnight;

[0049] 2) Washing: Add 200 μl of 1× PBST Buffer to each well and wash for 5 min. Pat dry on paper after washing. Change the paper after each patting. Wash the plate 3 times in total.

[0050] 3) Blocking: Prepare 1% BSA (dissolved in 1×PBS Buffer) blocking solution as needed. Pour it into a disposable sample loading tank. Use a pipette to add 200 μl to each well. Seal the plate with a sealing film and incubate it in a 37°C incubator for 2 hours. After completion, wash the plate in the same way as step 2);

[0051] 3) React with whole blood of patients with Plasmodium falciparum and normal human serum: dilute whole blood of patients with Plasmodium falciparum with 1×PBS Buffer at a ratio of 1:50, and dilute normal human serum with 1×PBS Buffer at a ratio of 1:100. Pipette the diluted samples into a 96-well plate with a pipette according to the principle of making a duplicate well for each sample in the same column, 100 μl per well, wherein the last two samples in each plate are added with normal human serum from Zhejiang Province as a negative control and 1×PBS Buffer as a blank control, respectively. After all are added, seal the plate with a sealing film and incubate in a 37°C constant temperature box for 1.5 hours. After the reaction is completed, wash the plate, and the washing process is the same as step 2);

[0052] 4) Reaction with goat anti-human IgG H&L (HRP conjugated): dilute with 1×PBS Buffer at a ratio of 1:10000, pour the diluted liquid into a disposable sample tank, pipette, add 100μl to each well, place in a 37℃ incubator and incubate for 1h. After the reaction is completed, wash the plate. The washing process is the same as step 2);

[0053] 5) Color development: Take out the soluble single-component TMB substrate solution from the 4°C refrigerator in advance and place it at room temperature. Keep it away from light until the temperature reaches room temperature. Pour a sufficient amount of solution into the disposable sample tank, pipette it, add 100 μl to each well and place it in a 37°C constant temperature box for color development. The color development time is about 5 to 10 minutes. Use a timer to determine the color development time.

[0054] 6) Stop color development: Pour the prepared stop solution (2M H2SO4) into the disposable sample tank for use in advance. Take it out from the 37°C incubator according to the color development. Immediately add 100 μl of stop solution to each well to stop the reaction.

[0055] 7) Use SYNERGY|H1 microplate reader (BioTek, Germany) to scan the 96-well plate at 450 nm, set the program (temperature 37°C, plate shaking for 5 s, absorbance at 450 nm) to read and export the data for storage.

[0056] 3. Data Statistical Analysis

[0057] Statistical data were calculated using mean ± standard deviation (X ± SD). The chi-square test was used to compare the positive reaction rate of whole blood of patients with Plasmodium falciparum and the false positive reaction rate of normal human serum. The chi-square test was used to compare the positive reaction rate of wild-type polypeptide and mutant polypeptide with whole blood of patients with Plasmodium falciparum. The independent sample z test was used to compare the OD values ​​of the wild-type polypeptide and mutant polypeptide with whole blood of patients with Plasmodium falciparum. The test level α = 0.05. The data results were statistically analyzed using Excel, and the statistical graphs were drawn using GraphPad Prism 8 software.

[0058] 4 Results

[0059] The mass spectra of the synthetic products of Plasmodium falciparum RIFIN polypeptides 8-RIFIN and 9-RIFIN are shown in Figure 1 As shown, the theoretical molecular weight of 8-RIFIN polypeptide is 2668.09, and the result of mass spectrometry analysis is 2667.3. The theoretical molecular weight of 9-RIFIN polypeptide is 2693.05, and the result of mass spectrometry analysis is 2692.4. The theoretical molecular weight is consistent with the molecular weight result of mass spectrometry analysis.

[0060] The results of antibody reaction between Plasmodium falciparum RIFIN polypeptide 8-RIFIN and 9-RIFIN and whole blood of patients with falciparum malaria and normal human serum are as follows Figure 2 As shown, the results showed that the antibody reactions of Plasmodium falciparum RIFIN polypeptides 8-RIFIN and 9-RIFIN with the whole blood of patients with falciparum malaria showed obvious differences. The antibody reaction positive rate of wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN to the whole blood of patients with falciparum malaria was significantly higher than that of mutant Plasmodium falciparum RIFIN polypeptide 9-RIFIN.

[0061] The results of chi-square test showed that the positive rate of immune reaction of wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN with whole blood of patients with falciparum malaria was 62.5% (75 / 120), and the false positive rate of immune reaction with normal human serum was 10.9% (5 / 46), which was significantly different (P<0.0001). The positive rate and false positive rate of immune reaction of mutant Plasmodium falciparum RIFIN polypeptide 9-RIFIN with the above two groups of samples were 29.2% (35 / 120) and 4.3% (2 / 46), respectively, which was also significantly different (P=0.0006). The above results show that the IgG antibody reaction of 8-RIFIN and 9-RIFIN in the whole blood of patients with falciparum malaria and the normal human serum were significantly different.

[0062] The chi-square test of the positive reaction rates of wild-type and mutant Plasmodium falciparum RIFIN polypeptides and whole blood of patients with falciparum malaria was performed for inter-group samples. It was found that the positive immune reaction rate of Plasmodium falciparum RIFIN polypeptide 8-RIFIN with whole blood of patients with falciparum malaria was significantly higher than that of mutant 9-RIFIN (P<0.0001), indicating that in terms of the reaction ability with whole blood (IgG) of patients with falciparum malaria, wild-type 8-RIFIN has a stronger immune reactivity, but due to amino acid mutations, the reaction ability of mutant 9-RIFIN with whole blood (IgG) of patients with falciparum malaria is greatly reduced.

[0063] Example 2

[0064] Binding reaction of Plasmodium falciparum RIFIN polypeptide 8-RIFIN with immunosuppressive receptor LILRB1

[0065] 1 Materials

[0066] 1.1 Plasmodium falciparum RIFIN polypeptides 8-RIFIN and 9-RIFIN were synthesized by GenScript Biotech Co., Ltd. (Nanjing).

[0067] 1.2 ELISA plate (96 wells) and sealing film were purchased from Thermo SCIENTIFIC.

[0068] 1.3 Main reagents and enzyme tools

[0069]

[0070]

[0071] 2 Methods

[0072] 2.1 Preparation of Plasmodium falciparum RIFIN polypeptide 8-RIFIN and 9-RIFIN working solution

[0073] 1) Preparation of peptide master solutions: Place 8-RIFIN and 9-RIFIN peptide tubes containing 1 mg of powder in a 4°C centrifuge and centrifuge at 12,000 rpm for 15 min. Then, according to the recommendations in the peptide synthesis report, 8-RIFIN is dissolved in 500 μl of ultrapure water to a concentration of 2 mg / ml, and 9-RIFIN is dissolved in 500 μl of dimethyl sulfoxide to a concentration of 2 mg / ml.

[0074] 2) Preparation of peptide working solution: dilute 8-RIFIN and 9-RIFIN stock solutions with coating buffer prepared in advance and placed in a 4°C refrigerator. Use coating buffer to dilute the peptide stock solutions in multiple ratios to prepare four working solutions with different concentration gradients, namely 1, 2, 4, and 8 μg / ml.

[0075] 2.2 Preparation of recombinant human LILLRB1 protein (Fc Chimera) working solution

[0076] 1) Preparation of recombinant human LILLRB1 protein (Fc Chimera) stock solution: Dissolve 100 μg of recombinant human LILLRB1 protein (Fc Chimera) powder in 250 μl of 1× PBS Buffer to prepare a solution with a concentration of 400 μg / ml;

[0077] 2) Preparation of recombinant human LILLRB1 protein (Fc Chimera) working solution: dilute the recombinant human LILLRB1 protein (Fc Chimera) stock solution with 1× PBS Buffer, 1:200, to prepare 2 μg / ml recombinant human LILLRB1 protein (Fc Chimera) working solution.

[0078] 2.3 ELISA of Plasmodium falciparum RIFIN polypeptides 8-RIFIN and 9-RIFIN with recombinant human LILLRB1 protein (Fc Chimera)

[0079] 1) Coating: Mark the peptide type on the side of the 96-well plate, add the prepared 1, 2, 4, 8 μg / ml peptide working solution to the corresponding wells of the 96-well plate at a volume of 100 μl / well, where the first two wells in each plate are 100 μl of coating solution as blank control, and seal the plate with a sealing film and place it steadily in a 4°C refrigerator overnight;

[0080] 4) Washing: Add 200 μl of 1× PBST Buffer to each well and wash for 5 min. Pat dry on paper after washing. Change paper after each patting. Wash the plate 3 times in total.

[0081] 5) Blocking: Prepare 1% BSA (dissolved in 1×PBS Buffer) blocking solution as needed. Pour it into a disposable sample loading tank. Use a pipette to add 200 μl to each well. Seal the plate with a sealing film and incubate it in a 37°C incubator for 2 hours. After completion, wash the plate in the same way as step 2);

[0082] 3) Binding reaction with recombinant human LILLRB1 protein (Fc Chimera): dilute the recombinant human LILLRB1 protein (Fc Chimera) stock solution with 1×PBS Buffer at a ratio of 1:200, and pipette the diluted sample into a 96-well plate, 100 μl per well. The last two samples in each plate are added with 1×PBS Buffer as negative controls. After all the samples are added, seal the plate with a sealing film and incubate in a 37°C incubator for 1 hour. After the reaction is completed, wash the plate. The washing process is the same as step 2);

[0083] 4) Reaction with goat anti-human IgG H&L (HRP conjugated): dilute with 1×PBS Buffer at a ratio of 1:10000, pour the diluted liquid into a disposable sample tank, pipette, add 100μl to each well, place in a 37℃ incubator and incubate for 1h. After the reaction is completed, wash the plate. The washing process is the same as step 2);

[0084] 5) Color development: Take out the soluble single-component TMB substrate solution from the 4°C refrigerator in advance and place it at room temperature. Keep it away from light until the temperature reaches room temperature. Pour a sufficient amount of solution into the disposable sample tank, pipette it, add 100 μl to each well and place it in a 37°C constant temperature box for color development. The color development time is about 5 to 10 minutes. Use a timer to determine the color development time.

[0085] 6) Stop color development: Pour the prepared stop solution (2M H2SO4) into the disposable sample tank for use in advance. Take it out from the 37°C incubator according to the color development. Immediately add 100 μl of stop solution to each well to stop the reaction.

[0086] 7) Use SYNERGY|H1 microplate reader (BioTek, Germany) to scan the 96-well plate at 450 nm, set the program (temperature 37°C, plate shaking for 5 s, absorbance at 450 nm) to read and export the data for storage.

[0087] 3. Data Statistical Analysis

[0088] The data were analyzed statistically using Excel, and the statistical graphs were drawn using GraphPad Prism 8 software.

[0089] 4 Results

[0090] The wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN and the mutant Plasmodium falciparum RIFIN polypeptide 9-RIFIN were respectively combined with the recombinant human LILLRB1 protein (Fc Chimera) for ELISA ( Figure 3 ), the results showed that the wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN can significantly react with the LILRB1 receptor. The formula of the fitting curve shows that R 2 =0.983; while its mutant 9-RIFIN hardly reacts with the inhibitory receptor LILRB1.

[0091] Example 3

[0092] Immunogenicity test of Plasmodium falciparum RIFIN peptide 8-RIFIN

[0093] 1 Materials

[0094] 1.1 Plasmodium falciparum RIFIN polypeptide 8-RIFIN was synthesized by GenScript Biotech Co., Ltd. (Nanjing).

[0095] 1.2 New Zealand rabbit polyclonal antibody was produced by Shanghai Youke Biotechnology Co., Ltd.

[0096] 1.3 ELISA plate (96 wells) and sealing film were purchased from Thermo SCIENTIFIC.

[0097] 1.4 Main reagents and enzyme tools

[0098] Reagent name Reagent Source 20×PBS Buffer Sangon Biotech Tween-20 Sangon Biotech Soluble one-component TMB substrate solution TIANGEN Goat anti-rabbit IgG H&L (HRP) Abcam BSA Solarbio Dimethyl sulfoxide,Analytical grade SIGMA <![CDATA[Na2CO3]]> Shanghai Jingxi Chemical <![CDATA[NaHCO3]]> Shanghai Jingxi Chemical <![CDATA[H2SO4]]> Sinopharm Other reagents All are domestic analytical reagents

[0099] 2 Methods

[0100] 2.1 Preparation of Plasmodium falciparum RIFIN polypeptide 8-RIFIN working solution

[0101] 1) Preparation of peptide master solution: Place the 8-RIFIN peptide tube containing 1 mg powder in a 4°C centrifuge and centrifuge at 12,000 rpm for 15 min. Then, according to the recommendations in the peptide synthesis report, dissolve 8-RIFIN in 500 μl ultrapure water to a concentration of 2 mg / ml.

[0102] 2) Prepare the peptide working solution: dilute the 8-RIFIN stock solution with the coating buffer prepared in advance and placed in a 4°C refrigerator, and dilute the peptide stock solution in multiples with the coating buffer to prepare a working solution with a concentration of 2 μg / ml.

[0103] 2.2 Preparation of rabbit serum polyclonal antibody working solution

[0104] 1) The immune New Zealand rabbit serum polyclonal antibody produced and provided by Shanghai Youke Biotechnology Co., Ltd., two New Zealand rabbits were immunized with the Plasmodium falciparum RIFIN polypeptide 8-RIFIN, and the rabbit serum polyclonal antibody was prepared by collecting blood before each immunization after four immunizations;

[0105] 2) Prepare New Zealand rabbit serum polyclonal antibody working solution: dilute New Zealand rabbit serum polyclonal antibody with 1× PBS Buffer to obtain working solutions with a dilution gradient of 1:1250, 1:2500, 1:5000, 1:10000 and 1:20000.

[0106] 2.3 Enzyme-linked immunosorbent assay of peptide 8-RIFIN and polyclonal antibody from immunized rabbit serum

[0107] 1) Coating: Mark the peptide type on the side of the 96-well plate, add the prepared 2 μg / ml peptide working solution to the corresponding wells of the 96-well plate at a volume of 100 μl / well, of which the first two wells in each plate are 100 μl of coating solution as blank control, and seal the plate with a sealing film and place it steadily in a 4°C refrigerator overnight;

[0108] 6) Washing: Add 200 μl of 1× PBST Buffer to each well and wash for 5 min. Pat dry on paper after washing. Change the paper after each patting. Wash the plate 3 times in total.

[0109] 7) Blocking: Prepare 1% BSA (dissolved in 1×PBS Buffer) blocking solution as needed. Pour it into a disposable sample loading tank and add 200 μl to each well using a pipette. Seal the plate with a sealing film and incubate it in a 37°C incubator for 2 hours. After completion, wash the plate in the same way as step 2);

[0110] 3) Binding reaction with immune New Zealand rabbit serum polyclonal antibody: New Zealand rabbit serum polyclonal antibody was diluted with 1×PBS Buffer according to the gradient of 1:1250, 1:2500, 1:5000, 1:10000 and 1:20000, and the diluted samples were pipetted into 96-well plates, 100 μl per well, and the last two samples in each plate were added with 1×PBS Buffer as negative control. After all the samples were added, the plates were sealed with sealing film and incubated in a 37°C incubator for 1 hour. After the reaction was completed, the plates were washed, and the washing process was the same as step 2);

[0111] 4) Reaction with goat anti-rabbit IgG H&L (HRP): dilute with 1×PBS Buffer at a ratio of 1:5000, pour the diluted liquid into a disposable sample tank, pipette, add 100 μl to each well, place in a 37°C incubator and incubate for 1 hour. After the reaction is completed, wash the plate. The washing process is the same as step 2);

[0112] 5) Color development: Take out the soluble single-component TMB substrate solution from the 4°C refrigerator in advance and place it at room temperature. Keep it away from light until the temperature reaches room temperature. Pour a sufficient amount of solution into the disposable sample tank, pipette it, add 100 μl to each well and place it in a 37°C constant temperature box for color development. The color development time is about 5 to 10 minutes. Use a timer to determine the color development time.

[0113] 6) Stop color development: Pour the prepared stop solution (2M H2SO4) into the disposable sample tank for use in advance. Take it out from the 37°C incubator according to the color development. Immediately add 100 μl of stop solution to each well to stop the reaction.

[0114] 7) Use SYNERGY|H1 microplate reader (BioTek, Germany) to scan the 96-well plate at 450 nm, set the program (temperature 37°C, plate shaking for 5 s, absorbance at 450 nm) to read and export the data for storage.

[0115] 3. Data Statistical Analysis

[0116] The data were analyzed statistically using Excel, and the statistical graphs were drawn using GraphPad Prism 8 software.

[0117] 4 Results

[0118] New Zealand rabbits were immunized with wild-type Plasmodium falciparum RIFIN polypeptide 8-RIFIN, and the obtained rabbit serum polyclonal antibodies were subjected to enzyme-linked immunosorbent assay with polypeptide 8-RIFIN. The results showed that the antibody reaction was significantly enhanced with the increase in the number of immunizations, and the serum polyclonal antibodies before the third and fourth immunizations were diluted to 10,000 times and still had a relatively obvious reaction. The above results show that 8-RIFIN has a strong enough immunogenicity to New Zealand rabbits.

[0119] The results of the above embodiments show that the present invention discloses a RIFIN polypeptide 8-RIFIN located at 243-267aa encoded by the RIFIN gene PF3D7_0900200 of Plasmodium falciparum. The function of 8-RIFIN is verified by antibody immune response and binding experiment with immunosuppressive receptor LILRB1 by ELISA method. Compared with the control mutant Plasmodium falciparum RIFIN polypeptide 9-RIFIN, the polypeptide of the present invention not only has a better ability to specifically recognize the whole blood of patients infected with falciparum malaria, but also can bind to the immunosuppressive receptor LILRB1, which indicates that the polypeptide of the present invention is the key region for the binding of Plasmodium falciparum RIFIN protein to LILRB1. Therefore, the present invention can be used as the basis for further studying the molecular mechanism of Plasmodium falciparum using RIFIN protein for immune escape, and has important application value in the elimination and control of malaria.

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Institute of Parasitic Diseases Control and Prevention (National Center for Tropical Diseases Research), Chinese Center for Disease Control and Prevention <120> A Plasmodium falciparum RIFIN polypeptide 8-RIFIN and its derivative products and applications <160> 2 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <400> 1 Tyr Gln Gly Ser Gln Cys Gly Ser Leu Ser Met Leu Leu Gly Lys Ser 1 5 10 15 Lys Pro Phe Cys Thr Phe Val Glu Gly 20 25 <210> 2 <211> 25 <212> PRT <213> Artificial Sequence <400> 2 Tyr Gln Gly Ser Gln Cys Gly Ser Leu Ser Met Leu Leu Gly Lys Ser 1 5 10 15 Asn Pro Tyr Cys Thr Leu Val Asn Glu 20 25

Claims

1. A Plasmodium falciparum RIFIN polypeptide 8-RIFIN, characterized in that The amino acid sequence of 8-RIFIN is shown in SEQ ID NO:

1.

2. Use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN according to claim 1 in preparing a kit for detecting Plasmodium falciparum.

3. The application according to claim 2, characterized in that: The detection sample of Plasmodium falciparum is whole blood or serum of human or animal.

4. A Plasmodium falciparum detection kit based on immunoassay technology, characterized in that: It comprises the Plasmodium falciparum RIFIN polypeptide 8-RIFIN as described in claim 1.

5. The Plasmodium falciparum detection kit according to claim 4, characterized in that: The immunoassay technique includes one or more of the following methods: enzyme-linked immunosorbent assay, immunoblotting, immunoenzyme staining, indirect fluorescent antibody detection and immunodiffusion.

6. The Plasmodium falciparum detection kit according to claim 5, characterized in that: When the detection is based on the enzyme-linked immunosorbent assay, the kit comprises a detection plate coated with the Plasmodium falciparum RIFIN polypeptide 8-RIFIN, a washing solution, an enzyme-labeled secondary antibody and a display solution.

7. Use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN according to claim 1 in the preparation of a vaccine for preventing and treating Plasmodium falciparum.

8. A vaccine for preventing and treating Plasmodium falciparum, characterized in that: The vaccine comprises the Plasmodium falciparum RIFIN polypeptide 8-RIFIN according to claim 1 and an adjuvant.

9. Use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN according to claim 1 in the preparation of a reagent that specifically binds to the immunosuppressive receptor LILRB1.

10. Use of the Plasmodium falciparum RIFIN polypeptide 8-RIFIN according to claim 1 in the preparation of a reagent or kit for blocking Plasmodium falciparum immune escape.

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