Babesia mocroti antigen proteins 2D33 and 2D36 and application of Babesia mocroti antigen proteins

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

Active Publication Date: 2019-02-22
FUDAN UNIV +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 mocroti antigen proteins 2D33 and 2D36 and application of Babesia mocroti antigen proteins
  • Babesia mocroti antigen proteins 2D33 and 2D36 and application of Babesia mocroti antigen proteins
  • Babesia mocroti antigen proteins 2D33 and 2D36 and application of Babesia mocroti antigen proteins

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 animal serum source

[0048] 1. Experimental animals and strains

[0049] The experimental animals were BALB / c mice aged 6-8 weeks, which were purchased from Shanghai Experimental Animal Research Center, Chinese Academy of Sciences.

[0050] The Babesia microti strain is Babesia microti PRA-99T, provided by the Institute of Experimental Zoology, Chinese Academy of Medical Sciences, was inoculated and preserved in liquid nitrogen for passage in this experiment.

[0051] 2. Serum collection

[0052] The worm blood containing the standard strain of Babesia microti taken out of liquid nitrogen was equilibrated to room temperature, diluted with sterile 0.9% saline at a ratio of 1:1 and mixed evenly. Draw 200 μl of diluted worm blood with a 1ml sterile syringe and inject it into the abdominal cavity of BALB / c mice to revive Babesia microti in the mice. Observing and counting the pathogens of Babesia microti by microscopic examination, before the parasitemia ...

Embodiment 2

[0054] Western blot hybridization and analysis identification of the crude protein of Babesia microti in embodiment 2

[0055] The Babesia microti strain is Babesia microti PRA-99T, provided by the Institute of Experimental Zoology, Chinese Academy of Medical Sciences. Babesia microti was inoculated into BALB / c mice, the whole blood was taken by anticoagulation, red blood cells were separated, lysed and centrifuged, and the precipitate was collected, which was determined to be Babesia worms by microscopic examination. By immunomics techniques such as figure 1 As shown, the crude protein of Babesia microti was hybridized with 7dpi and 30dpi mouse serum respectively, and by mass spectrometry analysis, 87 Babesia microti proteins were identified, including 8 by 7dpi mouse serum identified protein.

Embodiment 3

[0056] Example 3 Obtaining the Babesia microti antigen immune response spectrum

[0057] 1. Experimental materials and methods

[0058] 1.1 Vectors and strains

[0059] 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 site Xho I, BamH I) and Babesia microti genome DNA are provided by this experiment.

[0060] 1.2 Experimental Instruments and Reagents

[0061] (1) Main instruments:

[0062] PCR instrument; constant temperature metal bath; vertical laminar flow clean bench; full temperature shaking incubator; electric heating constant temperature incubator; desktop high-speed refrigerated centrifuge; small centrifuge; pot; decolorizing shaker; constant temperature mixer; 85-2 type constant temperature magnetic stirrer; nucleic acid electrophoresis instrument; protein electrophoresis system: BIO-RAD; gel imager: BIO-RAD; Microarray scanner.

[0063] (2) M...

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Abstract

The invention discloses Babesia mocroti antigen proteins 2D33 and 2D36 and application of the Babesia mocroti antigen proteins. The amino acid sequences of the Babesia mocroti antigen proteins are respectively represented by SEQ ID NO:4 or SEQ ID NO:6, and an encoding gene sequence of Babesia mocroti antigen proteins is represented by SEQ ID NO:3 or SEQ ID NO:5. An immunoreactions spectrum of Babesia mocroti is acquired, the immunoreactions of recombinant proteins Bm2D33 and Bm2D36 with mouse serum after an acute stage are obviously decreased in the protein chip analysis of specific IgG antibody, and the recombinant proteins Bm2D33 and Bm2D36 can be used for detecting disease transformation progress of Babesia mocroti from the acute stage to a chronic stage. The recombinant proteins Bm2D33and Bm2D36 present very high specificity, namely 100% in the cross reaction tests of the serum of patients with falciparum malaria.

Description

technical field [0001] The invention relates to the research fields of molecules, cells, proteomics and immunology, in particular to the preparation of the Babesia microti immune protein and the acquisition of immune response spectrum, as well as its application in the screening of candidate diagnostic antigens and immune protection vaccines. Background technique [0002] Babesia is a zoonotic parasitic disease caused by protozoa of the genus Babesia parasitizing in red blood cells, and is transmitted through tick bites and blood transfusions. There are more than 100 species of Babesia isolated from wild animals and domestic animals, among which the reported cases of infecting humans mainly include: B. microti and related pathogens, B. divergens and related pathogens, B. duncani and related pathogens, B. venatorum. In January 2011, Babesias was listed as a national notifiable infectious disease. In the United States, Babesia vole is responsible for most cases of Babesia vo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C07K14/44A61K39/018A61P33/02G01N33/569
CPCA61P33/02G01N33/56905C07K14/44A61K39/00G01N2469/10Y02A50/30
Inventor 胡薇徐斌刘秀凤周霞陈家旭程训佳陈军虎周晓农许学年张颋蔡玉春
Owner FUDAN UNIV
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