African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 4875

Full Length Research Paper

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

Pu Xue1,2, Hong-yu Han2, Ya-mei Jin2, Lian-lian Jiang2, Chun-lin Kong1,2, Hui Dong2, Qi-ping Zhao2, Ye Wang 2, Shun-hai Zhu 2, Ting Li 2, Fan-fan Shu2 and Bing Huang2*
1College of Life and Environment Sciences, Shanghai Normal University, Shanghai, 200234, China. 2Shanghai Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture, P. R. China
Email: [email protected]

  •  Accepted: 08 March 2013
  •  Published: 19 March 2013

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.

  • Articles on Google by: