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Babesia microti thioredoxin molecule and its gene and application

A technology of babesia thioredoxin and thioredoxin, applied in the field of bioengineering, can solve problems such as unstable effects

Active Publication Date: 2020-12-15
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, only a few drugs have been used for the treatment of babesia, and the effect is not stable. Therefore, the study of the infection and survival mechanism of babesia is the basis of drug target design

Method used

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  • Babesia microti thioredoxin molecule and its gene and application
  • Babesia microti thioredoxin molecule and its gene and application
  • Babesia microti thioredoxin molecule and its gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Gene Cloning and Sequence Analysis of Babesia microti thioredoxin molecule (BmTrx1)

[0028] 1. Materials and methods

[0029] 1.1. Parasites and experimental animals

[0030] Babesia microti was purchased from ATCCR PRA-99TM under the American Standard Culture Collection (ATCC), and was subcultured and preserved in Kunming mice in our laboratory.

[0031] 1.2. Bacteria and plasmids

[0032] Escherichia coli DH5α (Invitrogen) and Escherichia coli BL21(DE3) (Novagen) were used for plasmid construction and protein expression. The sequencing vector was pGEM-T Easy (Promega), and the expression vector was pET30a vector (Novagen).

[0033] 1.3. Molecular cloning of Babesia microti thioredoxin (BmTrx1)

[0034] The mouse erythrocytes infected with Babesia microti were collected, crushed, and the worms were collected by centrifugation. The parasite RNA was extracted with TRIzolreagent (Invitrogen), and then the genomic DNA of the parasite was removed with DNase ...

Embodiment 2

[0037] Example 2 Recombinant expression and purification of Babesia microti thioredoxin molecule (BmTrx1)

[0038] In order to reduce the influence of exogenous amino acids on the protease activity of BmTrx1, the target gene BmTrx1 was cloned into the expression vector pET30a, and only the His tag was introduced at the C-terminus, and then expressed in E.coli BL21.

[0039] With pET-Trx1-F:5'-AAGGAGATATA CATATGGTGAAAGAAGTACAAACTACTGCTGA-3' (SEQ ID NO. 8) and pET-Trx1-R: 5'-GGTGGTGGTG CTCGAG GACGTGCTTCTTGATAGTGCTCC-3'(SEQ ID NO.9) is the expression primer amplified to obtain the open reading frame sequence of BmTrx1, which is connected to the enzyme-digested (Nde I and Xho I) by In-Fusion HD Cloning Kit (Takara) in one step Expression vector pET-30a (Novagen), and transfected with DH5α. After the positive clones were obtained by sequencing, the positive plasmids were extracted and transfected into E.coli BL21(DE3) for protein expression. At 37°C, 1mMIPTG was induced for 8 ...

Embodiment 3

[0042] Example 3 Preparation of recombinant protein rBmTrx1 / His antiserum and detection by Western Blotting

[0043] After the recombinant protein rBmTrx1 / His was thoroughly mixed with an equal volume of Freund's complete adjuvant, about 100ug of protein per mouse was injected intraperitoneally. On the 14th day and 28th day after the initial immunization, booster immunization was carried out twice with fully mixed recombinant protein and incomplete adjuvant. The anti-rBmTrx1 / His serum of mice was collected after the antibody titer was determined by ELISA.

[0044] RBCs from mice infected with Babesia microti (2 to 10 days) and uninfected (0 days) were electrophoresed on a 15% SDS-PAGE gel, then transferred to Immobilon-P SQ membrane (Millipore). After blocking with 5% skimmed milk overnight at 4°C, the membrane was reacted with the collected anti-rBmTrx1 / His serum (diluted 1:200) at room temperature for 1 hour, and then washed 3 times with PBST, 10 minutes each time. After ...

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Abstract

The invention discloses an amino acid sequence of a Babesia microti thioredoxin molecule. The invention also discloses a Babesia microti thioredoxin molecule gene which comprises a nucleotide sequencefor coding an amino acid sequence as shown in SEQ ID NO.1. The Babesia microti thioredoxin molecule disclosed by the invention has an oxidation-resistant protective action on cells, and is suitable for screening thioredoxin inhibitor drugs for treating Babesia.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a thioredoxin molecule of Babesia microti and its gene and application. Background technique [0002] Babesia microti is a unicellular worm belonging to the Apicomplexa. Parasitic on domestic animals such as cattle and horses, pet dogs, wild animals and birds. Babesia vole can also infect humans, threatening the health of the population and food safety. Babesia parasitizes in the red blood cells of the host, divides and reproduces, produces toxins, and destroys red blood cells. Therefore, hemolytic anemia and hemoglobinuria occur after infection with babesia, and severe cases can lead to death. This pathogenic protozoan is distributed globally, mainly concentrated in Europe and the United States. In recent years, there have been reports of human infection with Babesia in Yunnan, Inner Mongolia and Taiwan. So far, only a few drugs have been used for the treatment of babe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/44C12N15/30C12N15/85C07K16/20C07K16/06G01N33/68G01N33/569A61K38/17A61K39/018A61K39/395A61P39/06A61P33/02
Inventor 龚海燕周金林张厚双曹杰周勇志海旭南赵少若
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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