Gene Cloning and Eukaryotic Expression of a Conserved Hypothetical Protein Gene CHP559 in Eimeria tenella

doi:10.13560/j.cnki.biotech.bull.1985.2015.07.024

Abstract

Abstract: The objective of this study is to construct eukaryotic recombinant expression plasmids of a conserved hypothetical protein gene(EtCHP559)of Eimeria tenella, and study the expression of EtCHP559 gene in transfected DF-1 cells. The full-length cDNA of EtCHP559 was cloned using 5'RACE approaches. Bioinformatics analysis revealed that the gene contained 1 746 bp, of which the ORF was 1 224 bp(position 104 - 1 327 bp)encoding 407 amino acids with molecular weight of 46 kD. The deduced amino acid sequence of the protein had a transmembrane region and signal peptide. The cDNA fragment consisting of complete ORF was amplified by RT-PCR, and ligated to the eukaryotic expression vector pcDNA3. 1-flag. The recombinant plasmid pcDNA3. 1-flag-EtCHP559 was identified successfully by PCR and restriction enzyme digestion, and the target band of approximate 1 224 bp was observed. Subsequently, the recombinant plasmid was transfected into DF-1 cells, Western blot recognized target protein of around 47 kD, and immunofluorescence also showed that green fluorescence in transfected DF-1 cells was observed. These results indicated that the full-length sequence of EtCHP559 was obtained, and the recombinant plasmid of EtCHP559 was successfully constructed and expressed in eukaryotic cells, which laid a foundation for the future research on functions of EtCHP559.

Key words: Eimeria tenella, CHP559 gene, DF-1 cell, eukaryotic expression

Zhai Qi, Huang Bing, Dong Hui, Zhao Qiping, Zhu Shunhai, Liang Siting, Li Sha, Yang Sihan, Han Hongyu. Gene Cloning and Eukaryotic Expression of a Conserved Hypothetical Protein Gene CHP559 in Eimeria tenella[J]. Biotechnology Bulletin, 2015, 31(7): 161-168.

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