Clone, expression and application of tender Eimeria tenella protein kinase (EtPK) gene

A technology of protein kinase gene and Eimeria coccidioides, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of safety, price and emergence of drug-resistant strains

Inactive Publication Date: 2009-11-11
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

At present, the main methods of controlling coccidiosis include chemical drug control and vaccine immunization prevention. However, due to the pr

Method used

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  • Clone, expression and application of tender Eimeria tenella protein kinase (EtPK) gene
  • Clone, expression and application of tender Eimeria tenella protein kinase (EtPK) gene
  • Clone, expression and application of tender Eimeria tenella protein kinase (EtPK) gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Cloning of the Eimeria tenella protein kinase (EtPK) gene:

[0096] 1. Materials

[0097] (1) Experimental animals

[0098] Luoman's high-quality yellow-feathered chickens are purchased from Shanghai Huizhong Breeding Chicken Farm. After hatching, they are transported back to the laboratory for breeding. The cages, feed, and drinking water are strictly sterilized.

[0099] (2) Experimental strains

[0100] Eimeria tenella: sporulated oocysts of Eimeria tenella, number: CAAS21111601, provided by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0101] (3) Main reagents

[0102] Trizol and GeneRaceTM Kit were purchased from Invitrogen; TaKaRa Agarose Gel DNA Purification Kit was purchased from Dalian Bao Biological Engineering Co., Ltd.; Agarose and PGEM-T-easy vector were purchased from Promega; JM109 competent cells were purchased from Dalian Bao Biological Co., Ltd.; ampicillin , IPTG were purchased from Huamei Biotechnology Co., L...

Embodiment 2

[0126] Expression of Eimeria tenella protein kinase (EtPK) gene in Escherichia coli

[0127] 1. Materials

[0128] (1) Main reagents

[0129] Restriction enzymes were purchased from Dalian Baobio Biotechnology Co., Ltd. T4 DNA ligase was purchased from Promega, and DNAMarker was purchased from Shanghai Meiji Biotechnology Co., Ltd.

[0130] (2) Plasmids and strains

[0131] The recombination cloning plasmid pGEM-T-EtPK is the above cloning recombination plasmid. Plasmids pGEX-4T-2, BL21(DE3) were provided by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0132] 2. Method

[0133] (1) Construction of recombinant expression plasmids:

[0134] The identified correct recombinant plasmid pGEM-T-EtPK, using the multiple cloning site of the cloning vector pGEM-T-easy and the expression vector pGEX-4T-2, selects the appropriate enzymes to carry out double digestion (Xho I and EcoR I) , EtPK and pGEX-4T-2 fragments were recovered after enzyme...

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Abstract

The invention discloses a novel tender Eimeria tenella protein kinase (EtPK) gene with a clone number of ZB1-A02 and the total length of 1684 bp, which contains an ORF with the length of 1527 bp and encodes 508 amino acids (a Genbank accession number is EF552216). The novel tender Eimeria tenella protein kinase (EtPK) gene is connected with a prokaryotic expression vector pGEX-4T-2 to establish a prokaryotic expression recombinant plasmid pGEX-4T-EtPK and is expressed in a colon bacillus system, and an expressed recombinant protein exists in a inclusion body way; a purified recombinant protein 4T-EtPK is processed by SDS-PAGE and then is transferred to a PVDF membrane, an antiserum of an E. tenella oocyst oral immune chicken is taken as first antibody, and a goat-anti chicken IgG is taken as second antibody to carry out Western-blot analysis, thereby the purified recombinant protein 4T-EtPK is shown to have certain antigenicity and can be used for developing chicken coccidiosis resistant medicament and preparing chicken coccidiosis resistant vaccines.

Description

Technical field: [0001] The invention relates to the fields of biotechnology medicine and gene technology, in particular to the cloning, expression and application of a novel Eimeria tenella protein kinase (EtPK) gene. Background technique: [0002] Chicken coccidiosis is a serious global parasitic disease caused by Eimeria parasitizing in the intestinal tract, causing huge economic losses to the chicken industry. At present, the main methods to control coccidiosis include chemical drug control and vaccine immunization prevention. However, due to the problems of safety, price and emergence of drug-resistant strains in these methods, it is urgent to find new control methods to control coccidiosis. The key to developing efficient and safe anti-coccidiosis genetically engineered vaccines and new drugs is to find important insect antigen genes. The present inventor takes Eimeria tenella (E.tenella), which is the most harmful to chickens, as the research object, and has carried ...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/10C12N15/70A61K38/45A61K39/00A61P33/02
Inventor 韩红玉黄兵林矫矫赵其平董辉姜连连颜彦姚倩樊玉娟李玉剑
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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