Construction of a yeast two-hybrid cDNA library from second-generation merozoites of Eimeria tenella

Abstract

To construct a yeast two-hybrid cDNA library from second-generation merozoites ofEimeria tenella (EtsMZ), total RNA of EtsMZ was extracted using Trizol reagent and mRNAs were isolated from total RNA. The first-strand cDNAs were synthesized by reverse transcription using MMLV. The dscDNAs acquired by Long-distance Polymerase Chain Reaction (LD-PCR) and purified by Chroma Spin TE-400 Column were transformed into AH109 yeast competent cells with pGADT7-Rec, a yeast expression vector, according to the screening method of yeast mating. All clones were harvested and the yeast two-hybrid cDNA library of the EtsMZ was constructed. The results show that the capacity and titer of the library were 3.22×1015 cfu and 8.05×1012 cfu/mL, respectively. PCR amplification revealed that the library contained approximately 96% recombinant clones, and the inserted cDNA fragments were between 300 and 2000 bp. In addition, five specific genes of E. tenella were amplified from the constructed cDNA library. It could be concluded that a yeast two-hybrid cDNA library of the EtsMZ was successfully constructed. The library can provide a foundation for screening invasion-related interaction proteins of E. tenella merozoite by yeast two-hybrid method. Key words: Coccidia, Eimeria tenella, second-generation merozoite, yeast two-hybrid, cDNA library.