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Echinococcus granulosus imago diagnosis protein gene and medical uses thereof

A technology of Echinococcus granulosus and gene, applied in the field of Echinococcus granulosus adult diagnostic protein gene and its medical use

Inactive Publication Date: 2015-11-11
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Fecal antigen detection methods mainly include arecoline catharsis method, ELISA method, PCR method, etc., but the response rate of arecoline catharsis method has certain limitations; ELISA method often has low sensitivity and is accompanied by certain cross-reactions ; PCR methods often have false positives, and have high requirements for instruments
There is no gold standard method for the detection of Echinococcus granulosus

Method used

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  • Echinococcus granulosus imago diagnosis protein gene and medical uses thereof
  • Echinococcus granulosus imago diagnosis protein gene and medical uses thereof
  • Echinococcus granulosus imago diagnosis protein gene and medical uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Screening, identification and prokaryotic expression of immune-related antigen genes

[0038] 1. Lambda phage plate culture

[0039] Take 30 μL of XL1-Blue strains and inoculate them into a test tube containing tetracycline LB liquid, and shake overnight at 37°C; take 4 mL of the bacterial solution and centrifuge at 5,000 g for 5 minutes, discard the supernatant, and add 200 μL of autoclaved 10 mM magnesium sulfate to resuspend the bacteria; add the resuspended Mix 200 μL of bacteria solution, 100 μL of 1×λBuffer and 100 μL of Echinococcus granulosus cDNA library, put in water bath at 37°C for 20 minutes; add LB / MgSO at 48°C 4 The top layer of agar was 3mL, mixed evenly and poured on the preheated LB plate and paved, after solidification, it was placed upside down in a 37°C incubator until needle-like phage plaques grew on the plate.

[0040] 2. Remove cross-antibody reactivity

[0041] Inoculate XL1-Blue in LB liquid medium containing tetracycline resistance and ...

Embodiment 2

[0066] Preparation of EdiagA864 Monoclonal Antibody and Polyclonal Antibody

[0067] 1. animal immunity

[0068] Take the EdiagA864 protein, add the same amount of adjuvant, fully emulsify it, inject it subcutaneously in multiple points on the back, and immunize female Balb / c mice aged 6-8 weeks. After three immunizations, the antibody titer of mouse serum was detected by indirect ELISA method, and the titer reached 200,000, and cell fusion was carried out 3 days later.

[0069] 2. cell fusion

[0070] The peritoneal cells of healthy mice were taken as feeder cells and spread on a 96-well cell culture plate, and the mouse myeloma cells and splenocytes of immunized mice were taken for cell fusion under the action of PEG.

[0071] 3. Screening and cloning of positive hybridoma cell lines

[0072] Cell culture supernatants on days 5 and 10 after fusion were collected for antibody detection. Hybridoma cell lines with positive test results were selected for cloning. Final...

Embodiment 3

[0082] Establishment of double-antibody sandwich ELISA method

[0083] 1. Horseradish peroxidase-labeled rabbit anti-EdiagA864 polyclonal antibody

[0084] The horseradish peroxidase purchased from Beijing Taitianhe Biotechnology Co., Ltd. was operated according to the instructions, and the rabbit anti-EdiagA864 polyclonal antibody was successfully labeled.

[0085] 2. Sample handling

[0086] Pretreatment of Echinococcus canis standard positive and negative samples: Weigh 1g of feces, add 5mL PBS (containing 0.3% Tween-20), mix well, ultrasonicate 3-4 times, pass through 3000g, centrifuge for 10min to take the supernatant .

[0087] 3. Detection of HRP-labeled antibody

[0088] After the standard positive and negative samples were treated, they were coated on the ELISA plate with the coating solution, blocked with 5% skimmed milk powder, and the HRP-labeled polyclonal antibody was diluted in different proportions as the secondary antibody. The reaction was terminated ...

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Abstract

The present invention provides an echinococcus granulosus imago diagnosis protein gene, further relates to a monoclonal antibody and a polyclonal antibody prepared by using the diagnosis gene protein so as to establish the canine echinococcus granulosus coproantigen detection method, further provides a protein gene for canine echinococcus granulosus ELISA detection and applications of the protein gene in establishment of the canine echinococcus granulosus double antibody sandwich ELISA method, and belongs to the technical field of canine disease detection.

Description

technical field [0001] The present invention provides a diagnostic protein gene of Echinococcus granulosus adults. In addition, the present invention also relates to the use of the diagnostic gene protein to prepare monoclonal antibodies and polyclonal antibodies, which can be used to establish a detection method for Echinococcus granulosus feces antigens Furthermore, it provides a protein gene for ELISA detection of Echinococcus granulosus in dogs, and is applied in the establishment of a double-antibody sandwich ELISA method for Echinococcus granulosus, belonging to the technical field of canine disease detection. Background technique [0002] Echinococcus granulosus (Echinococcus granulosus) Adult worms parasitize in the small intestine of dogs, causing loss of appetite, digestive disorders, and severe damage to the digestive system. Its eggs or proglottids can be excreted with dog feces and infect cattle, sheep, pigs and other animals as well as humans. So far, canine ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/70G01N33/68A61K39/00A61P33/10
Inventor 张西臣刘原源李建华宫鹏涛王晓岑安红燕杨举李赫张楠尹继刚
Owner JILIN UNIV
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