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Babesia voles 2d97 antigen protein and its application

A technology of antigenic protein and Babesia, applied in cell, proteomics and immunology research, and molecular fields, can solve the problems of unmonitored disease process, unclear antigen spectrum/immune response spectrum, and ineffective detection of samples

Active Publication Date: 2022-07-05
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 / immune response profile during the transition from the acute phase to the chronic phase of infection is still unclear, and the disease process is slow. less than monitoring

Method used

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  • Babesia voles 2d97 antigen protein and its application
  • Babesia voles 2d97 antigen protein and its application
  • Babesia voles 2d97 antigen protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Source of animal serum

[0046] 1. Experimental animals and strains

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

[0048] 2. Serum collection

[0049] The worm blood containing the standard strain of Babesia voles taken out from liquid nitrogen was equilibrated to room temperature, diluted 1:1 with sterile 0.9% normal saline and mixed well. Draw 200 μl of the diluted worm blood with a 1 ml sterile syringe and inject it into the abdominal cavity of BALB / c mice to revive Babesia voles in the mice. The pathogens of Babesia voles were observed and counted by microscopic examination. Before the parasemia reached the peak (no hemolysis occurred, t...

Embodiment 2

[0051] Example 2 Immunoblot hybridization and analysis and identification of crude protein of Babesia voles

[0052] Babesia strain Babesia microti PRA-99T, provided by the Institute of Laboratory Zoology, Chinese Academy of Medical Sciences. Babesia voles were inoculated into BALB / c mice, whole blood was collected after anticoagulation, erythrocytes were separated, lysed and centrifuged to collect the pellets, which were confirmed as Babesia worms by microscopic examination. Through immunomic techniques such as figure 1 As shown, the crude protein of Babesia vole was subjected to immunoblot hybridization with 7dpi and 30dpi mouse serum, respectively, and by mass spectrometry analysis, 87 Babesia vole proteins were identified, including 8 that were affected by 7dpi mouse serum. identified protein.

Embodiment 3

[0053] Example 3 Acquisition of Babesia Antigen Immune Response Spectrum of Voles

[0054] 1. Experimental materials and methods

[0055] 1.1 Vectors and strains

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

[0057] 1.2 Experimental instruments 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-trace UV / visible light spectrophotometer); Pot; destaining shaker; constant temperature mixer; 85-2 constant temperature magnetic stirrer; nucleic acid electrophoresis instrument; protein electro...

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Abstract

The invention discloses a Babesia vole 2D97 antigen protein and application thereof. Its amino acid sequence is shown in SEQ ID NO: 2, and its coding gene sequence is shown in SEQ ID NO: 1. The present invention obtains the immune response spectrum of Babesia voles, and finds that in the specific IgM antibody response, only an antigen protein 2D97 which exhibits a higher immune response with the early 3dpi and 7dpi mouse serum is screened, which can be used as an early diagnosis. Antigen candidates. The nucleotide sequence of the antigenic protein 2D97 was used for prokaryotic expression in Escherichia coli, and its recombinant protein was successfully obtained. In the protein chip analysis of specific IgM antibody, it showed that it was higher than the early 3,7dpi mouse serum. The immune response can be used for detection or diagnosis in the early and / or window period of Babesia voles infection 3 to 7 days.

Description

technical field [0001] The invention relates to the fields of molecular, cellular, proteomic and immunological research, in particular to the preparation of Babesia voles immune proteins, the acquisition of immune response profiles, and the application in screening of candidate diagnostic antigens and immune protection vaccines. Background technique [0002] Babesiosis is a zoonotic parasitic disease caused by the protozoa Babesia in red blood cells and is transmitted by tick bites and blood transfusions. There are more than 100 kinds of Babesia isolated from wild animals and livestock, among which 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, Babesiosis was listed as a national notifiable infectious disease. In the United States, the majority of Babesia cases are caused by Babesia vole, and it has been reported that between 1979 and 2...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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