柔嫩艾美耳球虫Cathepsin B基因的克隆表达及活性研究

doi:10.11843/j.issn.0366-6964.2015.10.017

Abstract

Abstract:

The aim of the present study was to clone the cathepsin B (Etcat B) gene involved in E.tenella using homologous method，and lay the the foundation for further gene expression and gene function study.Through analysis of E.tenella sequence published，the cDNA sequence of Etcat B was cloned by RT-PCR from unsporulated E.tenella Houghton strain.The Etcat B was recombined in trans-E1 prokaryotic expression vector.After sequence identification，the recombinant plasmid was transfected into E.coli rosetta to induce expression.The purified protein was used to immunize rabbits to produce polyclonal antibody，which was verified by ELISA，and was also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gelatin zymography.With the method of real-time PCR and Western blot，we detected the expression of Etcat B and Etcat L at different stage.Results showed that an 1 539 bp gene was obtained，which encoded 512 amino acid residues.The Etcat B cDNA fragment was sub-cloned into the prokaryotic expression vector trans-E1.After inducing in 1 mmol•L^-1 IPTG at 37 ℃ for 4 h，SDS-PAGE electrophoresis showed it was expressed mainly in the form of inclusion bodies；while at 4 ℃ overnight the protein was inducted into soluble expression.With the presence of reducing agent at low pH，the recombinant protein can degradate the gelatin，which proved that the recombinant enzyme having a certain activity.Etcathespin B was higher in the phase of gametophyte and merozoite，and gradually increased during sporulation process.Cloning and expression the cathepsin B in E.tenella，provides theoretical basis for further exploration of the role of the gene in chicken coccidia invasion and other biological functions.

CLC Number:

S852.723

LIU Ren-qiang，CAI Jian-ping，WANG Ming. Cloning，Expression and Activity of Cathepsin B in Eimeria tenella[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(10): 1838-1843.

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Cloning，Expression and Activity of Cathepsin B in Eimeria tenella

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