Babesia mocroti 2D97 antigen protein and application thereof

An antigen protein, Babesia technology, applied in the field of cell, proteomics and immunology, molecular field, can solve the problem of unmonitored disease process, unclear antigen spectrum/immune response spectrum, ineffective detection of samples, etc.

Active Publication Date: 2018-11-27
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the infection process of babesiosis, samples from the early stage of infection (window period) cannot be effectively detected, and the antigen profile/imm

Method used

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  • Babesia mocroti 2D97 antigen protein and application thereof
  • Babesia mocroti 2D97 antigen protein and application thereof
  • Babesia mocroti 2D97 antigen protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Source of animal serum

[0046] 1. Laboratory animals and insect strains

[0047] The experimental animals were BALB / c mice aged 6-8 weeks, purchased from the Shanghai Experimental Animal Research Center of the Chinese Academy of Sciences. Babesia microti PRA-99T, provided by the Institute of Experimental Zoology, Chinese Academy of Medical Sciences, was inoculated and stored in liquid nitrogen for passages in this experiment.

[0048] 2. Serum collection

[0049] The blood of the standard strain of Babesia voles taken from the liquid nitrogen is balanced to room temperature, and diluted with sterile 0.9% normal saline at a ratio of 1:1 and mixed evenly. Use a 1ml sterile syringe to draw 200μl of diluted worm blood and inject it into the abdominal cavity of BALB / c mice to resuscitate Babesia voles in the mice. Observe and count the pathogen of Babesia voles by microscopic examination. Before the worm disease reaches the peak (no hemolysis, the infection rate of red...

Embodiment 2

[0051] Example 2 Western blot hybridization, analysis and identification of Babesia voles crude protein

[0052] Babesia microti PRA-99T, provided by the Institute of Experimental Zoology, Chinese Academy of Medical Sciences. The voles Babesia was inoculated into BALB / c mice, and whole blood was collected for anticoagulation, red blood cells were separated, lysed, centrifuged, and the precipitate was collected and confirmed to be Babesia by microscopic examination. Through immunoomics techniques, such as figure 1 As shown, the crude protein of Babesia voles was hybridized with 7dpi and 30dpi mouse sera, respectively, and 87 Babesia voles protein was identified by mass spectrometry analysis, including 8 sera from 7dpi mice. The identified protein.

Embodiment 3

[0053] Example 3 Obtaining the antigen immune response spectrum of Babesia voles

[0054] 1. Experimental materials and methods

[0055] 1.1 Vectors and strains

[0056] E. coli DH5α was purchased from Beijing Tiangen Biochemical Technology Co., Ltd. The linearized vector pEU-E01-His-TEV-MCS-N2 (restriction endonuclease cut sites Xho I, BamH I) and Babesia voles genomic DNA were preserved and provided in this experiment.

[0057] 1.2 Experimental equipment and reagents

[0058] (1) Main instruments:

[0059] PCR instrument; constant temperature metal bath; vertical laminar flow clean bench; full temperature shaking incubator; electric heating constant temperature incubator; desktop high-speed refrigerated centrifugation; small centrifuge; NanoDrop2000 (ultra-micro ultraviolet / visible light spectrophotometer); high-pressure steam sterilization Pot; Decoloring shaker; Constant temperature mixer; Model 85-2 constant temperature magnetic stirrer; Nucleic acid electrophoresis instrument; Pr...

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Abstract

The invention discloses a babesia mocroti 2D97 antigen protein and application thereof. The protein has an amino acid sequence of SEQ ID NO:2 as shown in the specification, and has an encoded gene sequence of SEQ ID NO:1 as shown in the specification. An immunoreaction spectrum of babesia mocroti is obtained, results show that in a specific IgG antibody reaction, only one antigen protein 2D97 which has relatively intense immunoreactions with early-stage mouse serum of 3dpi and 7dpi is screened, and the antigen protein can be a candidate for early-stage antigen diagnosis. Escherichia coli prokaryotic expression is carried out with the nucleotide sequence of the antigen protein 2D97, a recombinant protein of escherichia coli is successfully obtained, and the protein has relatively intense immunoreactions with early-stage mouse serum of 3dpi and 7dpi in protein chip analysis of a specific IgM antibody, and can be applied to detection or diagnosis at an early stage and/or window stage within 3-7 days after infection of babesia mocroti.

Description

Technical field [0001] The invention relates to the research fields of molecules, cells, proteomics and immunology, in particular to the preparation of Babesia vole immune protein and the acquisition of immune response spectrum, as well as the application in screening candidate diagnostic antigens and immune protection vaccines. Background technique [0002] Babesia is a zoonotic parasitic disease caused by Babesia protozoa parasitic in red blood cells. It is transmitted through tick bites and blood transfusions. There are more than one hundred kinds of Babesia isolated from wild animals and domestic animals. Among them, the reported cases of human infection mainly include: B.microti and related pathogens, B.divergens and related pathogens, B.duncani and related pathogens, B. venatorum. In January 2011, Babesia was listed as a national statutory infectious disease. In the United States, most Babesia cases are caused by Babesia voles. It has been reported that from 1979 to 2009,...

Claims

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Application Information

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IPC IPC(8): C12N15/30C07K14/44C07K16/20C12Q1/6893G01N33/68A61K39/018A61P33/02
CPCA61K39/018A61P33/02G01N33/68C12Q1/6893C07K14/44C07K16/20A61K2039/552
Inventor 徐斌胡薇刘秀凤周霞黄继磊张颋莫筱瑾陈家旭陈军虎程训佳
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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