Polypeptide for detecting asymptomatic infection of malaria of plasmodium vivax and application of polypeptide

Abstract

The invention belongs to the technical field of molecular biological detection, and relates to a polypeptide for detecting asymptomatic infection of plasmodium vivax malaria and application. The polypeptide for detecting the asymptomatic infection of the malaria of the plasmodium vivax is an amino acid sequence as shown in SEQ ID No.1, or a homologous sequence which is subjected to substitution, deletion or insertion of one or more amino acids or has the sequence similarity greater than 75%. The polypeptide has an amino acid sequence as shown in SEQ ID No. 1. The polypeptide SEQ ID No.1 in the invention specifically detects asymptomatic tertian malaria infection, and can effectively deal with tertian malaria input threats in the Burmese border area. Meanwhile, 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 asymptomatic malaria infection caused by Plasmodium vivax and its application. Background Technology

[0002] Malaria caused by Plasmodium vivax is difficult to eradicate globally due to the lack of efficient and accurate detection tools. Existing detection tools struggle to detect Plasmodium vivax infections characterized by asymptomatic infection. The life cycle of Plasmodium vivax indicates that it forms dormant cells in the human liver, remaining dormant for an unpredictable period before entering the bloodstream and causing infection. During this dormant period, the parasite is absent or present in small quantities in the blood, resulting in asymptomatic infection, which is difficult to monitor and treat promptly. Furthermore, detection efforts have revealed that asymptomatic infected individuals can still transmit malaria, highlighting the need to address the issue of asymptomatic infection. Summary of the Invention

[0003] The purpose of this invention is to provide a polypeptide for detecting asymptomatic malaria infection 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 asymptomatic infection with Plasmodium vivax malaria, wherein the polypeptide is the amino acid sequence shown in SEQ ID No. 1, or a homologous sequence with one or more amino acid substitutions, deletions, or insertions, or sequence similarity greater than 75%.

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

[0006] An application of the aforementioned polypeptide in the detection of Plasmodium vivax malaria.

[0007] Application of the peptide in the detection of asymptomatic Plasmodium vivax malaria.

[0008] Application of the polypeptide in detecting asymptomatic Plasmodium vivax malaria in the China-Myanmar border region.

[0009] A method for detecting asymptomatic Plasmodium vivax malaria involves diluting a blood sample and adding it dropwise into a test kit containing the peptide. The peptide reacts with the antigen-antibody binding of the blood sample to determine whether the sample is infected with asymptomatic Plasmodium vivax malaria.

[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: In regions where vivax malaria has been eliminated (such as the China-Myanmar border region where local malaria has been eliminated and only imported malaria remains), the impact of asymptomatic infection is more significant compared to areas with severe endemic malaria. To consolidate the malaria elimination achievements in these regions and completely eradicate malaria, it is necessary to improve detection efficiency and develop new detection tools. The peptide in SEQ ID No. 1 of this invention specifically detects asymptomatic vivax malaria infection and can effectively address the threat of imported vivax malaria in the China-Myanmar border region.

[0012] Meanwhile, the peptides in this invention can be encapsulated in rapid detection kits, which are characterized by portability, high efficiency, and low cost, making them suitable for on-site sample testing in malaria-endemic areas. Attached Figure Description Figure 1 This is a B-cell epitope prediction diagram of the polypeptide SEQ ID No. 1 in the merozoite surface protein (MSP9) of Plasmodium vivax provided in an embodiment of the present invention.

[0013] Figure 2 Computer molecular docking prediction simulation of the polypeptide SEQ ID No.1 provided in the embodiments of the present invention.

[0014] Figure 3 The ELISA results of the polypeptide SEQ ID No.1 provided in this embodiment of the invention show the results of symptomatic infection samples (SP, n=74) and asymptomatic infection samples (ASP, n=20) of Plasmodium vivax in the China-Myanmar border region compared with normal control samples (NC, n=96).

[0015] Figure 4 The ROC curve results of the sensitivity / specificity analysis of the peptides provided in the embodiments of the present invention include statistical analysis of symptomatic and asymptomatic infected samples.

[0016] Figure 5 The chi-square test results for the SP and ASP groups of the peptide provided in this embodiment of the invention are shown below. The SP group includes 28 positive data points and 46 negative data points, while the ASP group includes 15 positive data points and 5 negative data points. The chi-square test result is a chi-square value (χ²) of 8.761 and a p-value of 0.0031 (<0.05). This indicates a significant difference in the distribution of positive / negative results between the SP and ASP groups. Detailed Implementation

[0017] 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.

[0018] 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.

[0019] Example 1 The key sites for binding of the hematogenous protein MSP9 of Plasmodium vivax to human blood antibodies (i.e., B-cell epitopes in the MSP9 protein) 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 ).

[0020] Table 1: Basic protein information of Plasmodium vivax protein MSP9, including protein number, abbreviation, and amino acid length; amino acid sequence, position, length, and IEDB prediction score of the polypeptide of SEQ ID No.1.

[0021] 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.

[0022] Depend on Figure 2 As can be seen, the obtained peptide has one binding site with the antibody, and the binding energy is low (a low binding energy indicates a tight binding).

[0023] After prediction, the polypeptide was chemically synthesized and prepared by Hubei Qiangyao Biotechnology Co., Ltd. SEQ ID No. 1: KKMFPK.

[0024] 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 (the threshold for this epitope is 0.089). Infected samples with OD data higher than the threshold are serologically positive samples, and those with OD data lower than the threshold are negative samples.

[0025] Depend on Figure 3 It can be seen that the 94 individual samples infected with Plasmodium vivax were divided into symptomatic samples (n=74) and asymptomatic samples (n=20) based on the presence or absence of clinical symptoms. The light intensity (OD) of the asymptomatic group was significantly higher than that of the symptomatic group, and the seropositivity rate was higher (37.84% in the symptomatic group and 75% in the asymptomatic group).

[0026] The sensitivity and specificity of the peptide were analyzed using ROC curve analysis of the above detection results. The AUC value was 0.586 in the symptomatic group and 0.956 in the asymptomatic group, indicating that SEQ ID No. 1 has a higher discriminant value for asymptomatic infection. Figure 4 (and Table 2).

[0027] Table 2: Grouping of SEQ ID No.1 peptide and comparison of AUC values ​​of each group.

[0028] Then, a chi-square test was used to compare the positive / negative data of symptomatic and asymptomatic positive samples using peptides. The results showed a significant difference (p<0.05). ),Right now Figure 5 The proportion of positive samples in the asymptomatic (ASP) group was significantly higher than that in the symptomatic (SP) group, as shown in Table 3. Figure 5 (and Table 3).

[0029] Table 3: Statistical analysis of positive and negative data in each group in the chi-square test.

[0030] The above data analysis shows that the polypeptide in SEQ ID No. 1 of this invention can efficiently detect asymptomatic clinical infection with Plasmodium vivax in the China-Myanmar border region, and has high sensitivity / specificity discrimination ability.

Claims

1. A polypeptide for detecting asymptomatic infection of Plasmodium vivax malaria, characterized by: The polypeptide is the amino acid sequence shown in SEQ ID No. 1, or a homologous sequence with one or more substitutions, deletions or insertions of amino acids or a sequence similarity of more than 75%.

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

1.

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

4. Use of a polypeptide according to claim 3, characterized in that: The polypeptide is used for detecting asymptomatic Plasmodium vivax malaria.

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

6. A method of detecting asymptomatic Plasmodium vivax malaria, characterized by: The blood sample to be detected is diluted and added dropwise into a detection kit containing the polypeptide of claim 1, and the antigen-antibody binding reaction between the polypeptide and the sample blood is determined by the enzyme-linked immunosorbent assay (ELISA) to determine whether the sample is an asymptomatic Plasmodium vivax malaria infection sample.

7. The method of detecting asymptomatic Plasmodium vivax malaria according to claim 6, characterized in that: The antigen-antibody binding reaction between the polypeptide and the sample blood is determined by the enzyme-linked immunosorbent assay (ELISA), and if the antigen-antibody binding is effective, i.e., the optical density (OD) intensity is higher than the threshold value, it is confirmed that the sample is an individual infected with Plasmodium vivax malaria. If there is no significant antigen-antibody binding, i.e., the optical density (OD) intensity is lower than the threshold value, it is confirmed that the sample is an individual not infected with Plasmodium vivax malaria.

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