Polypeptide fragment specifically binding to serum of Babesiosis patients, and amino acid sequence thereof

A technology of polypeptide fragments and Babesia, which is applied in the fields of biomedicine and clinical applications, can solve the problems of complex preparation process, unstable activity, and high cost, improve specificity and sensitivity, and overcome missed detection of parasites in blood smears The effect of misdiagnosis

Active Publication Date: 2018-12-18
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, BMSA is prepared by worm purification and Escherichia coli recombinant expression. The preparation process is complicated, the cost is high, and the activity is unstable. In addition, part of the amino acid sequence in the full-length BMSA protein has been confirmed to contain no antigenic epitopes recognized by patient serum. Therefore, using the full-length BMSA protein as a serum diagnostic antigen is prone to cross-reaction

Method used

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  • Polypeptide fragment specifically binding to serum of Babesiosis patients, and amino acid sequence thereof
  • Polypeptide fragment specifically binding to serum of Babesiosis patients, and amino acid sequence thereof
  • Polypeptide fragment specifically binding to serum of Babesiosis patients, and amino acid sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 The biological characteristics of Babesia microti merozoite surface antigen BMSA

[0021] According to the Babesia microti merozoite surface antigen BMSA gene sequence (such as figure 1 Shown) The deduced protein is composed of 326 amino acids (as shown in Sequence 2), and the size is 35.117kDa; the isoelectric point pI is predicted to be 5.21 by ExPAS software, and the 302nd amino acid is predicted by the PredGPI software to be the GPI anchor site, SignalP4 .1 The software predicts that the N-terminal 24 amino acids are signal peptides (such as figure 2 shown); TMPred software predicts the transmembrane segment from extracellular to intracellular, from amino acid 310 to 326 (Score2066) (eg image 3 shown), the PotParam software predicts that negatively charged amino acids account for 53%, and the proportion of positively charged amino acids is 43%; homology modeling predicts the three-dimensional structure of BMSA as Figure 4 shown.

Embodiment 2

[0022] Example 2 Epitope Analysis and Verification of the Babesia vole merozoite surface antigen BMSA

[0023] According to the Disco Tope 2.0 software, there are 6 antigenic epitopes (as shown in Table 1) for the B-cell epitope of the Babesia vole merozoite surface antigen BMSA, and the peptide synthesis of these 6 epitopes is carried out by artificial peptide synthesis. Synthesize; then screen and identify the epitope by indirect ELISA method through the serum of mice infected with Babesia, the method is as follows: Add 100 μL / well Coating Buffer (0.5 μg of artificially synthesized BMSA protein polypeptide) to the 96-well microtiter plate , in a wet box, coated overnight at 4°C; remove the coating solution, wash the plate with PBST, add 1% skimmed milk-PBS at room temperature, and block for 1 hour; add double-diluted Babesia-infected mouse serum and BMSA After immunization, mouse serum was 100 μL / well (1:32001:6400, diluted in PBS), incubated at room temperature for 1 h in a...

Embodiment 3

[0026] Embodiment 3 enzyme-linked immunosorbent assay (ELISA) detects babesiosis patient's serum

[0027] Add 100 μL / well Coating Buffer (0.5 μg artificially synthesized BMSA protein polypeptide) to the 96-well ELISA plate, and coat overnight at 4°C in a wet box; remove the coating solution, wash the plate with PBST, and add 1% skimmed milk respectively - PBS at room temperature, block for 1 hour; add 100 μl of healthy or patient serum (diluted 1:400) to each well, and incubate for 1 hour at room temperature in a humid box with healthy human serum (diluted 1:400) as negative control. After washing the plate with PBS (containing 0.05% Tween20), 100 μl of horseradish peroxidase-labeled goat anti-human IgG (diluted 1:1000) was added to each well, and incubated for 1 h at room temperature in a humid chamber. After washing the plate with PBS (containing 0.05% Tween20), add 200 μl / well of chromogenic solution, react in the dark for 30 minutes at room temperature, and finally add 2M ...

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Abstract

The invention belongs to the technical field of biomedicine and clinical application, relates to a polypeptide fragment specifically binding to the serum of Babesiosis patients, and an amino acid sequence thereof. According to the present invention, a series polypeptide fragments are obtained by analyzing the structure and the epitope of a Babesia merozoite surface protein BMASA, and the polypeptide fragment is prepared through polypeptide synthesis, and can be used for preparing the serological immunodiagnostic reagent for Babesiosis; and with the polypeptide fragment, the polypide missing diagnosis and the polypide misdiagnosis of the blood smear in the diagnosis of Babesiosis can be overcome, and the specificity and the sensitivity of the diagnosis of Babesiosis can be significantly improved.

Description

technical field [0001] The invention relates to the technical field of biomedicine and clinical application, in particular to a polypeptide fragment specifically binding to the serum of Babesia patients, its amino acid sequence and its application. Background technique [0002] The prior art discloses that Babesiosis (Babesiosis) is a new zoonotic disease caused by the infection of the protozoa that parasitizes in red blood cells——Babesia. Studies have shown that the disease is mainly transmitted through blood sucking after the arthropod tick bites the host, and can also be transmitted through blood transfusion and mother-to-child transmission. Clinical practice shows that after Babesia infection, according to the host's own immune status and species, it may manifest as asymptomatic, malaria-like symptoms and dangerous symptoms; the general symptoms of Babesia infection in immune-competent persons are fatigue, fever, Headache, chills, night sweats, anemia, loss of appetite,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/44G01N33/68G01N33/569
CPCG01N33/56905G01N33/6854C07K14/44G01N2469/20
Inventor 程训佳满素勤付永锋
Owner FUDAN UNIV
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