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Application of Echinococcus granulosus glutaredoxin‑1

A technology for Echinococcus granulosus and Echinococcus granulosus disease, which is applied in the application field of Echinococcus granulosus glutaredoxin-1, can solve the problems of complex composition, unstable antigen source, difficult purification and the like, and achieves The effect of high specificity and sensitivity, good diagnostic performance

Active Publication Date: 2018-01-23
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on the immunodiagnosis of echinococcosis granulosus mainly focuses on the antigen of crude cyst fluid, but its composition is complex, and it has defects such as difficulty in large-scale purification, unstable source of antigen, high cost, and low diagnostic specificity.
Lack of standard validated specific recombinant antigens is an unsolved problem in the immunodiagnosis of echinococcosis

Method used

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  • Application of Echinococcus granulosus glutaredoxin‑1
  • Application of Echinococcus granulosus glutaredoxin‑1
  • Application of Echinococcus granulosus glutaredoxin‑1

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1: Extraction of total RNA from Echinococcus granulosus

[0044] The midtaen stage larvae of Echinococcus granulosus (Echinococcus granulosus) cysts came from naturally infected sheep livers. The samples were collected from Xining, Qinghai or Ganzi, Sichuan, and stored in liquid nitrogen.

[0045] Take out the Echinococcus granulosus preserved in liquid nitrogen, grind it in a mortar, and then extract the total RNA according to the instructions of the animal tissue RNA extraction kit of Tiangen.

[0046] (1) Add 300μL of lysis solution for every 10-20mg of protocephalic larvae, and grind with a grinding rod;

[0047] (2) Add Proteinase K (10μL) and RNase-Free ddH2O (590μL) to the homogenate, mix and react for 15min (56°C).

[0048] (3) Centrifuge for 5 min (12,000 rpm) and transfer the supernatant to a clean tube; add 0.5 times the volume of the supernatant to the tube, mix and transfer to the adsorption column, centrifuge for 1 min (12,000 rpm) Discard the waste liquid....

Embodiment 2

[0055] Example 2: Synthesis of the first strand cDNA

[0056] Use the extracted total Echinococcus granulosus RNA as the template, Oligo dT(18) as the reverse transcription primer, and refer to the Thermo Company reverse transcription kit instructions for operation:

[0057] (1) Prepare the reaction mixture on ice

[0058] Oligo dT 18 1μL

[0059] Template RNA 1μL

[0060] DEPC ddH 2 O 1μL

[0061] (2) After incubating at 70°C (5min), transfer to ice to cool

[0062] (3) Add the following reactants in order on ice:

[0063] 5reaction buffer 4μL

[0064] Ribonuclease inhibitor 1μL

[0065] 10dNTP mix 2μL

[0066] (4) Add RevertAid after incubating at 37°C (5min) TM 1μL of reverse transcriptase.

[0067] (5) PCR program: 42°C, 1h; 70°C, 10min.

[0068] (6) The obtained cDNA is stored at -80°C.

Embodiment 3

[0069] Example 3: Amplification of Eg-Grx1 gene

[0070] According to the gene sequence of Eg-Grx1 (EgrG_000124800) published in GeneDB (http: / / www.genedb.org / Homepage / Egranulosus), primers were designed with Primer Premier 5.0 software:

[0071] Upstream: 5’-CC GGAATTC ATGTGGCGCTTTTTATC-3’ underlined as EcoR I

[0072] Downstream: 5’-CCG CTCGAG CTCTAAAAGTTCAGCAAGTG-3’ underscore Xho I

[0073] Amplification system (25μL): DNA template 1μL each, upstream and downstream primers 1μL each, PCR Mixture 12.5μL, sterilized double distilled water 9.5μL.

[0074] Amplification conditions: pre-denaturation: 95°C for 5min; 38 cycles (denaturation: 95°C, 40s; annealing: 54°C, 45s; extension: 72°C, 45s); final extension: 72°C, 10min.

[0075] Using the cDNA of Echinococcus granulosus protocercaria as a template, a 351bp band was amplified ( figure 1 ).

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Abstract

The invention relates to the technical field of biology, and concretely discloses application of echinococcus granulosa glutaredoxin-1 to preparation of an immunizing antigen and / or a kit for detecting echinococcosis granulose. When being used as an immunizing antigen, the echinococcus granulosa glutaredoxin-1 can be recognized by sheep positive blood serum naturally infecting the echinococcosis granulose. When being applied to indirect ELISA detection, the echinococcus granulosa glutaredoxin-1 has high specificity and sensitivity; the clinic detection coincidence rate is as high as 97.9 percent. The detection method built by using the echinococcus granulosa glutaredoxin-1 as the immunizing antigen has a good diagnosis effect, and can be used for the primary screening of the echinococcus granulosa of sheep in an infected area.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more specifically to the application of Echinococcus granulosus glutaredoxin-1. Background technique [0002] Echinococcus granulosus (Echinococcus granulosus) belongs to the genus Taeniidae (Echinococcus), in which the tape stage larvae parasitize the liver, lung and other organs of animals and humans and cause Echinococcus granulosus. Cystic echinococcosis is a zoonotic disease that is mainly parasitic in humans and domestic animals and other mammals. More than 90% of Echinococcus granulosus cysts grow in the host's liver, lungs, or both. Echinococcus granulosus has a worldwide distribution, causing a series of economic and public health problems. In some endemic areas, the infection rate can be as high as 5-10%, and the mortality rate can be as high as 2-4%. It is estimated that human cystic echinococcosis loses at least 1-3.6 million Disability Adjusted Life Years (DALYs) each year, whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 杨光友宋星桔
Owner SICHUAN AGRI UNIV
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