E.tenella TERT associated protein gene and medical uses thereof

A technology of Eimeria cocci and related proteins, applied in the field of genetic engineering, can solve problems such as proteins and functions that have not yet been reported.

Inactive Publication Date: 2015-09-30
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports about E. tenella TERT-related proteins and their functions

Method used

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  • E.tenella TERT associated protein gene and medical uses thereof
  • E.tenella TERT associated protein gene and medical uses thereof
  • E.tenella TERT associated protein gene and medical uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] E. tenella Construction of TRBD bait expression plasmid

[0032] 1. Materials

[0033] Trizol (Invitrogen); reverse transcriptase kit (Tiangen); T4 DNA ligase, Ex Taq, dNTPs, pMD18-T, Eco R I, Bam H I (TaKaRa).

[0034] 2. Methods and results

[0035] 1 E. tenella Extraction of total RNA from sporulated oocysts

[0036] 1.1 Treatment of utensils: After washing the mortar and pestle, soak them in 0.1% DEPC-H 2 overnight in O. Then wrap it in tin foil and dry bake at 180°C for 3 hours for later use;

[0037] 1.2 Collected E. tenella Grind sporulated oocysts with liquid nitrogen for 20 minutes, add 1 mL Trizol, and lyse at room temperature for 5 minutes;

[0038] 1.3 Add 200 μL of chloroform, mix well, place at room temperature for 15 minutes, and centrifuge at 12000 g, 4°C for 15 minutes;

[0039] 1.4 Transfer about 600 μL RNA from the upper layer to a new centrifuge tube, add 500 μL isopropanol, mix well, place at room temperature for...

Embodiment 2

[0058] Detection of Toxicity and Self-activation of Recombinant Bait Protein on Yeast Strains

[0059] 1. Materials

[0060] X-α-GAL, salmon sperm DNA (Sigma); yeast nitrogen source (Biosharp); c-Myc (Epitomics); ECL chemiluminescence kit (Thermo); yeast plasmid mini-prep kit (Tiangen).

[0061] 2. Methods and results

[0062] 1 Preparation of Y187 competent yeast and transformation of recombinant bait plasmid pGBKT7-TRBD

[0063] 1.1 Streak the cryopreserved Y187 cell plate and culture in a 30°C incubator for 3-4 days until a single colony with a diameter of 2-3 mm grows;

[0064] 1.2 Pick Y187 yeast monoclonal cells, put them into a 50mL Erlenmeyer flask containing 10mL YPDA liquid medium, and blow evenly, 30°C, 210r / min shaking culture to OD 600 >1.5; take 4~5mL of yeast into 50mL YPDA liquid medium, culture at 30°C, shake at 210r / min until OD 600 =0.4~0.6;

[0065] 1.3 Collect the yeast liquid in a 50mL centrifuge tube, centrifuge at 700g for...

Embodiment 3

[0083] Screening of TRBD Interacting Proteins

[0084] 1. Materials

[0085] -Trp / -His / -Leu DO Supplement, -Trp / -His / -Leu / -Ade DO Supplement (Clontech).

[0086] 2. Methods and results

[0087] 1 Screening of proteins interacting with TRBD in yeast two-hybrid cDNA library

[0088] 1.1 Pick the Y187 monoclonal transformed into pGBKT7-TRBD in 50mL SD / -Trp liquid medium;

[0089] 1.2 30°C, 270r / min shaker culture to OD 600 >0.8 (16~20h), centrifuge at room temperature at 700g for 5min, discard the supernatant;

[0090] 1.3 Resuspend the bottom and count with a cell counting plate to adjust the cell concentration to ≥1×10 8 cells / mL;

[0091] 1.4 Mix 5mL bait bacteria Y187 and 1mL library AH109 cells (≥2×10 7 cells) in a 1L sterile Erlenmeyer flask;

[0092] 1.5 Add 45mL 2×YPDA liquid medium (containing Kan + 50μg / mL), the library tube was washed twice with 1mL 2×YPDA, and the washing solution was added to the Erlenmeyer flask;

[0093] 1.6 30 ℃ constant temperature s...

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Abstract

The present invention provides an E.tenella TERT associated protein gene, and particularly relates to applications of the protein in telomerase activity regulation, wherein the protein has a negative regulation effect on the E.tenella telomerase activity, and develops the new research field for deep research on cell and molecular biology characteristics of coccidian and finding of new chicken coccidian preventing and treating drugs, vaccine acting target sites and the like. According to the present invention, the advantages of high efficiency and practicality are provided, a plurality of positive interaction proteins can be obtained through the yeast two-hybrid screening and verification so as to easily find the new function protein, and the method of the present invention is mature and is easy to operate compared with the comparative analysis, the proteomics, the transcriptomics, and other technologies; and the E.tenella virus transforming vector-mediated ribozyme provides the simple and easy-performing method for the research on the functions of the gene in the insect body.

Description

technical field [0001] The present invention provides a kind of Eimeria tenella TERT related protein gene, particularly relate to the effect of this protein in the regulation of telomerase activity, this protein is to E. tenella The telomerase activity has a negative regulating effect, may become a potential target for controlling coccidiosis, and belongs to the technical field of genetic engineering. Background technique [0002] Chicken coccidiosis is a parasitic disease that is distributed worldwide and seriously endangers the development of the chicken industry. Eimeria, as an intracellular parasite, parasitizes in the chicken intestine and causes chicken coccidiosis, which is mainly characterized by intestinal lesions, and seriously endangers the development of the poultry industry. At present, the control of coccidiosis mainly depends on the use of anticoccidiosis drugs and vaccines. However, due to the lack of detailed understanding of coccidiosis cells and molecula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C12N15/10C12N15/70A61K39/012A61P33/02
Inventor 张西臣赵娜宫鹏涛李建华武斌王旭吴娜杨举李赫张楠杨正涛
Owner JILIN UNIV
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