Cloning, expression and application of eimeria tenella protein disulfide isomerase gene

A technology of disulfide bond isomerase and Eimeria coccidia, which is applied in application, gene therapy, genetic engineering, etc., can solve the problems of safety, price and emergence of drug-resistant strains

Inactive Publication Date: 2009-04-29
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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

Method used

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  • Cloning, expression and application of eimeria tenella protein disulfide isomerase gene
  • Cloning, expression and application of eimeria tenella protein disulfide isomerase gene
  • Cloning, expression and application of eimeria tenella protein disulfide isomerase gene

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Experimental program
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Embodiment 1

[0099] Cloning of a protein disulfide isomerase (EtPDI) gene from Eimeria tenella:

[0100] 1. Materials

[0101] (1) Experimental animals

[0102] 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.

[0103] (2) Experimental strains

[0104] Eimeria tenella: sporulated oocysts of Eimeria tenella, number: CAAS 21111601, provided by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0105] (3) Main reagents

[0106] Trizol and GeneRaceTM Kit were purchased from Invitrogen; TaKaRa Agarose Gel DNAPurification 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 Bio...

Embodiment 2

[0130] Expression of Eimeria tenella protein disulfide isomerase (EtPDI) gene in Escherichia coli

[0131] 1. Materials

[0132] (1) Main reagents

[0133] Restriction enzymes were purchased from Dalian Baobio Biotechnology Co., Ltd. T4 DNA ligase was purchased from Promega, and DNA Marker was purchased from Shanghai Meiji Biotechnology Co., Ltd.

[0134] (2) Plasmids and strains

[0135] The recombination cloning plasmid pGEM-T-EtPDI is the above cloning recombination plasmid. Plasmids pGEX-4T-2 and BL21(DE3) were provided by our institute.

[0136] 2. Method

[0137] (1) Construction of recombinant expression plasmids:

[0138] With the identified correct recombinant plasmid pGEM-T-EtPDI, use the multiple cloning site of the cloning vector pGEM-T-easy and the expression vector pGEX-4T-2, select the appropriate enzyme to carry out double digestion (Bam H I and EcoR I) , BW1-E06 and pGEX-4T-2 fragments were recovered after enzyme digestion, and the recombinant expressio...

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Abstract

The invention discloses an E.tenella protein disulfide linkage isomerase gene EtPDI (Clone ID is BW1-E06,and the Genbank accession number of is EF552214). The gene is connected with a procaryon expression vector pGEX-4T-2; a procaryon expression recombination plasmid pGEX-4T-EtPDI is constructed and is expressed in a colibacillus system; and most of the expressed recombining protein exists in a soluble form. The recombining protein 4T-EtPDI is purified to carry out SPS-PAGE and is transferred to a PVDF film; and antiserum of E.tenella oocyst oral immunized chicken is used as first resistance and goat anti-chicken IgG is used as second resistance to carry out Western-blot analysis, thereby indicating that the gene has certain antigen. The gene is used for preparing an anti-chicken coccidiosis drug and an anti-chicken coccidiosis vaccine.

Description

Technical field: [0001] The invention relates to the field of biotechnology medicine and gene technology, in particular to the cloning, expression and application of the Eimeria tenella new protein disulfide bond isomerase gene (EtPDI) 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 has Eimeria tenella (E.tenella), which is the most harmful to chickens, as the research obj...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/09A61K48/00A61P33/00
Inventor 黄兵韩红玉林矫矫赵其平董辉姜连连王鑫韩静芳张建哲卞庆松闫晓菲
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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