Polypeptide for detecting malaria symptom infection of plasmodium vivax and application

Abstract

The invention belongs to the technical field of molecular biological detection, and relates to a polypeptide for detecting malaria symptom infection of plasmodium vivax and application. The polypeptide is any one or more amino acid sequences as shown in SEQ ID No. 1 to SEQ ID No. 4. The polypeptide can be coated in a detection tool of a rapid detection kit, and the tool has the characteristics of portability, high efficiency, low cost and the like, and is suitable for field sample detection in a malaria prevalence area.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, and relates to a polypeptide for detecting malaria symptoms caused by Plasmodium vivax and its application. Background Technology

[0002] Malaria caused by Plasmodium vivax is a global infectious disease. Infection with Plasmodium vivax can lead to clinical symptoms such as fever, chills, dizziness, and headache. Current detection tools for Plasmodium vivax are insufficient for continuous monitoring of malaria infection, and related peptide-coated rapid detection kits are relatively rare, resulting in virtually no application of these kits in the China-Myanmar border region. Summary of the Invention

[0003] The purpose of this invention is to provide a polypeptide for detecting malaria symptoms caused by Plasmodium vivax and its application.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: A polypeptide for detecting malaria symptoms of Plasmodium vivax infection, wherein the polypeptide is any one or more of the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 4.

[0005] The polypeptide has the amino acid sequence shown in SEQ ID No. 1 to SEQ ID No. 4.

[0006] The The amino acid sequence of SEQ ID No. 1 is SSQMLRGSV; The amino acid sequence of SEQ ID No. 2 is IYYNPHT; The amino acid sequence of SEQ ID No. 3 is VDEAAYV; The amino acid sequence of SEQ ID No.4 is EIKENNQVVI.

[0007] An application of the aforementioned polypeptide in the detection of Plasmodium vivax malaria infection.

[0008] The application of the polypeptide in detecting Plasmodium vivax malaria infection in the China-Myanmar border region.

[0009] A method for detecting Plasmodium vivax malaria symptoms: (1) Coating one or more of the polypeptides shown in SEQ ID No. 1-SEQ ID No. 4 into a rapid detection kit; (2) Obtain a small amount of blood sample to be tested, dilute it, and add it to the test kit; (3) The sample is determined to be a Plasmodium vivax malaria infection sample by the binding reaction of the polypeptide with the antigen and antibody of the blood sample and by the intensity of the optical density (OD) in the enzyme-linked immunosorbent assay.

[0010] The polypeptide reacts with the antigen and antibody of the blood sample. If the antigen and antibody binding is effective, that is, the optical density (OD) intensity in the enzyme-linked immunosorbent assay is higher than the threshold, it is confirmed that the sample is infected with Plasmodium vivax malaria. If there is no significant binding between the antigen and antibody, i.e., the optical density (OD) intensity is below the threshold, the individual is confirmed as an uninfected sample of Plasmodium vivax malaria.

[0011] Advantages of this invention: The peptides SEQ ID No. 1-SEQ ID No. 4 of this invention are specifically designed for the detection of vivax malaria infection. These peptides can be encapsulated in rapid diagnostic kits, which are portable, efficient, and low-cost, making them suitable for field sample testing in malaria-endemic areas. Attached Figure Description

[0012] Figure 1 The above is a B-cell epitope prediction diagram of the peptides in the merozoite surface protein (MSP9) and apical membrane antigen 1 (AMA1) of Plasmodium vivax provided in the embodiments of the present invention. In the present invention, SEQ ID No.1-SEQ ID No.3 belong to MSP9 protein and SEQ ID No.4 belongs to AMA1 protein, which are marked in the figure respectively.

[0013] Figure 2 The computer molecular docking prediction simulations of each polypeptide provided in the embodiments of the present invention are as follows: A is SEQ ID No. 1, B is SEQ ID No. 2, C is SEQ ID No. 3, and D is SEQ ID No. 4.

[0014] Figure 3 The ELISA experimental results of each polypeptide provided in the embodiments of the present invention show the results of Plasmodium vivax infection samples (PV, n=95) in the China-Myanmar border area compared with normal control samples (NC, n=95).

[0015] Figure 4 The ROC curve results for sensitivity / specificity analysis of each peptide provided in the embodiments of the present invention. Detailed Implementation

[0016] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0017] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; the reagents used in the embodiments are commercially available products.

[0018] Example 1 The key sites for binding of the hematogenous proteins MSP9 or AMA1 of Plasmodium vivax to human blood antibodies (i.e., B-cell epitopes in the MSP9 or AMA1 proteins) were predicted using the IEDB database in immunobioinformatics. During prediction, secondary or tertiary structure scoring data of existing amino acids in the IEDB database were used to obtain scores for each amino acid site in the full sequence of the MSP9 or AMA1 protein. Amino acid sequences with scores not lower than 0.5 were selected as potential B-cell epitope sites, resulting in the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 4 (see [link to relevant documentation]). Figure 1 ).

[0019] Table 1: Basic protein information of Plasmodium vivax protein MSP9 / AMA1, including protein number, abbreviation, and amino acid length; amino acid sequence, position, length, and IEDB prediction score of polypeptides SEQ ID No.1-SEQ ID No.4.

[0020] The amino acid sequences obtained through the above predictions were then used to simulate possible binding sites between peptides and antibodies using an immunobioinformatics molecular docking prediction tool. Figure 2 Specifically: Using the existing tertiary structure data of MSP9 / AMA1 protein and IgG antibody in the PDB database, the ClusPro / zDock molecular docking prediction software was used to predict and obtain the binding model with the best binding effect.

[0021] Depend on Figure 2 It is evident that all obtained peptides have at least one binding site with the antibody, and the binding energy is low (low binding energy indicates tight binding).

[0022] The predicted peptides were prepared by Hubei Qiangyao Biotechnology Co., Ltd. through chemical synthesis, as shown in Table 1.

[0023] Example 2 The obtained peptides were validated using clinical serum samples, specifically as follows: Enzyme-linked immunosorbent assay (ELISA) was used. Clinical serum samples included 94 individuals infected with Plasmodium vivax from Yingjiang County, a region on the my country-Myanmar border, and 96 normal control samples. In the ELISA experiment, four amino acid sequences were detected separately. The polypeptide protein was coated onto a 96-well plate (incubated at 37°C for 1.5 hours), bound to the serum sample (incubated at 37°C for 2 hours), and then bound to rabbit anti-human IgG secondary antibody (incubated at 37°C for 1 hour). The binding effect was visualized using TMB developing solution, and the light intensity (OD450) data was obtained by reading the average OD of the normal control samples +2. The standard deviation of OD is used to obtain the threshold of the sequence. Infected samples with OD data above the threshold are serologically positive samples, and those below the threshold are negative samples.

[0024] Depend on Figure 3 As shown in Table 2, threshold values ​​for MSP9 and AMA1 proteins were obtained from healthy control samples using positive control peptides. The threshold for MSP9 was 0.089, and for AMA1 it was 0.076 (Table 2). Clinical serum samples of *Plasmodium vivax* were tested using peptides SEQ ID No. 1-SEQ ID No. 4. Samples with values ​​higher than the threshold values ​​for each protein were considered positive. The positive rates (number of positive samples above the threshold / total number of samples) for the four peptides were 61.70%, 70.21%, 90.43%, and 77.66%, respectively. Combining the positive rate results of the four peptides, the overall positive rate reached 97.87% (92 / 94).

[0025] Table 2: Positive control sequences of Plasmodium vivax protein MSP9 / AMA1 and their threshold calculation process and results. Cut-off = mean OD + 2 OD standard deviation.

[0026] The sensitivity and specificity of each peptide were calculated based on the above detection results using threshold values, and the results are shown in Table 3. ROC curve analysis was then used to analyze the sensitivity and specificity of each peptide, and the AUC values ​​were 0.898, 0.802, 0.980, and 0.910, respectively. Figure 4 ).

[0027] Table 3: Number of positive samples, positive rate, sensitivity, specificity and AUC value of peptides SEQ ID No.1-SEQ ID No.4.

[0028] The above data analysis shows that the four peptides in this invention can efficiently detect clinical infection of Plasmodium vivax in the China-Myanmar border region, and have high sensitivity and specificity.

Claims

1. A polypeptide for detecting an infection with symptoms of malaria by Plasmodium vivax, characterized in that: The polypeptide is any one or several of the amino acid sequences shown in SEQ ID No. 1-SEQ ID No.

4.

2. The polypeptide for detecting P. vivax malaria infection according to claim 1, characterized in that: The polypeptide is the amino acid sequence shown in SEQ ID No. 1-SEQ ID No.

4.

3. The polypeptide for detecting P. vivax malaria infection according to claim 2, characterized in that: The amino acid sequence of SEQ ID No. 1 is SSQMLRGSV. The amino acid sequence of SEQ ID No. 2 is IYYNPHT. The amino acid sequence of SEQ ID No. 3 is VDEAAYV. The amino acid sequence of SEQ ID No. 4 is EIKENNQVVI.

4. Use of a polypeptide according to claim 1, characterized in that: The polypeptide is used for detecting Plasmodium vivax malaria infection.

5. Use of a polypeptide according to claim 4, characterized in that: The polypeptide is used for detecting Plasmodium vivax malaria infection in the border area between China and Myanmar.

6. A method for detecting Plasmodium vivax malaria infection, characterized in that: (1) any one or several of the polypeptides shown in SEQ ID No. 1-SEQ ID No. 4 are coated in a rapid detection kit; (2) a small amount of sample blood to be detected is diluted and added to the detection kit; (3) the antigen-antibody binding reaction of the polypeptide and the sample blood is determined by the strength of the optical density (OD) in the enzyme-linked immunosorbent assay to determine whether the sample is a Plasmodium vivax malaria infection sample.

7. The method of detecting P. vivax malaria infection according to claim 5, characterized in that: The antigen-antibody binding reaction of the polypeptide and the sample blood, if the antigen-antibody binding is effective, that is, the optical density (OD) intensity in the enzyme-linked immunosorbent assay is higher than the threshold value, it is confirmed as a Plasmodium vivax malaria infection sample individual; If there is no significant antigen-antibody binding, that is, the optical density (OD) intensity is lower than the threshold value, it is confirmed as a Plasmodium vivax malaria non-infection sample individual.

Images