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Babesia microti thioredoxin reductase molecule, and gene and applications thereof

A technology of babesia thioredoxin and thioredoxin, applied in the field of bioengineering

Inactive Publication Date: 2017-06-06
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

For a long time, the Trx system of Plasmodium falciparum has been studied extensively, but the research in Babesia microti has not been reported so far. Therefore, in order to find drug targets, it is necessary to study the characteristics of Babesia microti TrxR in order to design drugs targeting reasonable inhibitor

Method used

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  • Babesia microti thioredoxin reductase molecule, and gene and applications thereof
  • Babesia microti thioredoxin reductase molecule, and gene and applications thereof
  • Babesia microti thioredoxin reductase molecule, and gene and applications thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Gene Cloning and Sequence Analysis of Babesia microti thioredoxin reductase molecule (BmiTrxR)

[0035] 1. Materials and methods

[0036] 1.1. Parasites

[0037] Babesia vole is quoted from the American Type Culture Collection (ATCC), numbered ATCC R PRA-99TM was inoculated and preserved in Kunming mice in our laboratory.

[0038] 1.2. Bacteria and plasmids

[0039] Escherichia coli Top 10 cells (Invitrogen) and Escherichia coli BL21(DE3) (Novagen) were used for plasmid construction and protein expression. The cloning and sequencing vector used pGEM-T Easy (Promega), and the expression vector used pET28a vector (Novagen).

[0040] 1.3. Molecular cloning of Babesia microti thioredoxin reductase (B.microti TrxR)

[0041] TRIzol reagent (Invitrogen) was used to extract the RNA of merozoites, and then the RNA was digested with DNase I (TOYOBO) to remove the genomic DNA. cDNA was obtained from total RNA of parasites by reverse transcription, and the whole st...

Embodiment 2

[0044] Example 2 Recombinant expression and purification of Babesia microti thioredoxin reductase molecule (BmiTrxR)

[0045] The full-length BmiTrxR gene was double-digested by NcoI and XhoI restriction sites, then subcloned into the expression vector pET-28a (Novagen), and sequenced to ensure the accuracy of the sequence. The full-length gene was expressed in E.coliBL21(DE3) by constructing a His-tagged protein. The expression bacteria were induced by 1mM IPTG, and after incubation at 16°C for about 12 hours, the cells were collected and stored at -80°C. Recombinant protein through Ni 2+ Purified with affinity resin (Novagen), the induced cells were resuspended with binding buffer (NI-NTA Buffer Kit, Novagen), then ultrasonically disrupted, centrifuged at 12,000×g, 4°C for 10 min, and the soluble supernatant was loaded into the purification resin, then washed twice with washing buffer, and finally the target protein was eluted with elution buffer (NI-NTA Buffer Kit, Novage...

Embodiment 3

[0047] Preparation of embodiment 3 recombinant protein BmiTrxR antiserum and Western Blotting detection

[0048] After the purified recombinant protein is mixed with complete adjuvant, mice are immunized by intraperitoneal injection (about 200g recombinant protein each), and then these mice are boosted once every two weeks, twice in total, and the recombinant protein needs to be used Incomplete adjuvant premix. Mouse blood samples were collected by orbital bleeding, and antibody titers were detected by ELISA.

[0049] Mouse red blood cells infected with Babesia microti and uninfected mouse red blood cells were mixed with equal amount of 2×SDS gel-loading buffer, and then boiled for 10 minutes to denature the protein. After gel electrophoresis on 12% SDS-PAGE, it was transferred to NC membrane and blocked overnight at 4°C with 5% skimmed milk. The blocked NC membrane was incubated with recombinant protein antiserum (diluted 1:200) at room temperature for 1 hour, and then wash...

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Abstract

The invention discloses an amino acid sequence of a babesia microti thioredoxin reductase molecule. The invention also discloses a gene of the babesia microti thioredoxin reductase molecule, and the gene includes a nucleotide sequence encoding the amino acid sequence shown as SEQ ID NO.1. The babesia microti thioredoxin reductase molecule has good reduction activity for DTNB, insulin, a substrate BmiTrx and direct-line homologous protein EcoTrx, and is suitable for screening a BmiTrx inhibitor medicine treating babesiosis.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Babesia microti thioredoxin reductase molecule and its gene and application. Background technique [0002] Babesia microti is a new zoonotic protozoan disease transmitted by ticks. It parasitizes in the red blood cells of human and rodent hosts, causing symptoms such as fever, anemia, and systemic exhaustion. The elderly, The infection is most severe and can lead to death in patients who have had their spleens removed or who are immunocompromised. Since the first case of human infection with Babesia microti was detected in California, USA in 1969, thousands of cases of human infection have been reported, and there is an increasing trend in recent years. Babesia vole is mainly prevalent in the United States and some European countries, and has been reported in other parts of the world, such as Japan, South Korea, South Africa, Brazil, India and other countries and Taiwan...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12Q1/26A61K38/44A61P33/02
CPCA61K38/00C12N9/0051C12Q1/26C12Y108/01009
Inventor 周金林赵少若龚海燕张厚双曹杰周勇志
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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