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Eimeria tenella heat shock protein 60 gene as well as cloning, expression and application thereof

A technology of Eimeria and heat shock protein, 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: 2011-03-23
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 problems of safety, price and emergence of drug-resistant strains in these methods, it is urgent to find new control methods to control coccidiosis

Method used

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  • Eimeria tenella heat shock protein 60 gene as well as cloning, expression and application thereof
  • Eimeria tenella heat shock protein 60 gene as well as cloning, expression and application thereof
  • Eimeria tenella heat shock protein 60 gene as well as cloning, expression and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Cloning of Eimeria tenella heat shock protein 60 (Et HSP60) gene:

[0120] 1. Material

[0121] (1) Experimental animals

[0122] Roman high-quality yellow-feather chickens were purchased from Shanghai Huizhong breeder farm and transported back to the laboratory for breeding after the shells were hatched. The cages, feed, and drinking water were all strictly disinfected.

[0123] (2) Experimental strain

[0124] Eimeria tenella: Eimeria tenella sporulated oocysts, number: CAAS21111601, preserved and provided by the Shanghai Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0125] (3) Main reagents

[0126] 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; TOP10 competent cells were purchased from Dalian Bao Biological Co., Ltd.; Cana Penicillin and IPTG were purchased from Huamei Bioe...

Embodiment 2

[0151] Expression of Eimeria tenella heat shock protein 60 (EtHSP60) gene in Escherichia coli

[0152] 1. Material

[0153] (1) Main reagents

[0154] Restriction endonucleases were purchased from Dalian Bao Biological Biotechnology Co., Ltd. T 4 DNA ligase was purchased from Promega, and DNA Marker and standard protein molecular weight were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0155] (2) Plasmids and strains

[0156] The recombinant cloning plasmid pGEM-T-Et HSP60 is the above cloned recombinant plasmid. Plasmids pET28a(+) and BL21(DE3) were provided by Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences.

[0157] 2. Method

[0158] (1) Construction of recombinant expression plasmid:

[0159] The identified correct recombinant plasmid pET28a(+)-Et HSP, using the cloning vector pGEM-T-easy and the multiple cloning site of the expression vector pET28a(+), select appropriate enzymes for double digestion (BamH I and Not I) ). Af...

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Abstract

The invention discloses a new Eimeria tenella heat shock protein 60 (Et HSP60) gene. The cloning number is ZB2-D07, the total length is 1802 bp, and the Et HSP60 gene contains a 1455 bp complete open reading frame (ORF) and can code 484 amino acids (Genbank accession number: FJ911605). The gene is connected with a prokaryotic expression vector pET28a(+) to construct a prokaryotic expression recombinant plasmid pET28a(+)-Et HSP60 and is expressed in an escherichia coli system. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is carried out by purifying the recombinant protein pET28a(+)-Et HSP60, and then the recombinant protein pET28a(+)-Et HSP60 is transferred to a PVDF (Polyvinylidene Fluoride) membrane. The antiserum of immuned chicken orally taking E. tenella oocyst is taken as a primary antibody, and goat-anti-chicken IgG (immunoglobulin G) is taken as a secondary antibody to carry out Western-blot analysis. The analysis result shows that the recombinant protein pET28a(+)-Et HSP60 has certain antigenicity and can be used for preparing a medicine for resisting chicken coccidiosis and a vaccine resisting chicken coccidiosis.

Description

Technical field [0001] The invention relates to biotechnology, in particular to the cloning, expression and application of a new E. tenella heat shock protein (HSPs) 60 (Et HSP60) gene. Background technique [0002] Chicken coccidiosis is a serious global parasitic disease caused by Eimeria parasitizing in the intestines, causing huge economic losses to the chicken industry. At present, the main methods of controlling coccidiosis include chemical drug control and vaccine immunization prevention. However, due to the safety, price and emergence of drug-resistant strains of these methods, there is an urgent need to find new prevention methods to control coccidiosis. The key to the development of highly efficient and safe genetically engineered vaccines and new drugs against coccidiosis lies in the search for parasite antigen genes. The inventors took Eimeria tenella (E. tenella), which is the most serious damage to chickens, as the research object, and carried out researches on the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C12P19/34C12N15/70C12P21/02A61K39/012A61P33/02C12R1/19C12R1/90
Inventor 韩红玉黄兵颜彦赵其平董辉姜连连周杰李洋郭涛平宪卿
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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