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
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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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