Construction And Characterization Of The Yeast Two-Hybrid CDNA Library Of Duck Embryo-fibroblast

Gosling plague is caused by Goose parvovirus. It is a kind of acute or subacute septicemia infection diseases, causes domestic goslings and Muscovy duckings infected under the age of20days. The disease spread fast, with high incidence and its mortality can reach to90%-100%. Gosling plague still be one of the most jeopardized contagious disease. GPV can culture on DEF or GEF, also can cause CPE after injected3-5days. We can observe syncytium and acidophilia inclusion body in cells by staining with HE. The yeast two-hybrid system is an effective method for the study of protein-protein, protein-micromolecule interaction. We constructed the cDNA library of the DEF, we will find the cell receptor in the library with yeast two-hybrid. It is very effective for GPV’s prevention and manipulation.The DEF was infected with GPV can occur CPE, that is to say there are GPV’s cell receptor on DEF. In the research, first we make the DEF, when the morphosis of the DEF was good and uniform, we extract the total RNA. We construct the yeast two-hybrid cDNA library of DEF with the method of SMART. The procedures including:the first-strand synthesis, amplification of ds cDNA by long distance PCR, Column purification of ds cDNA with a CHROMA SPIN TE-400column, preparation of Competent Yeast Cells, transformation of Competent Yeast Cells with ds cDNA and PGADT7. two-hybrid library screening and identifying. Library construction takes place directly in our library yeast strain Y187, utilizing the highly potent homologous recombination machinery of Saccbaromyces cerevisiae, then screen the positive colonies on SD/-Leu plates, calculate the titer and the transformation efficiency. Finally we examined how long the inserted fragment of the ds cDNA by colony PCR.The DEF occurred obviously CPE after infecting GPV. We examined the result of the total RNA of the DEF by ultraviolet spectrophotometer and1%agarose gel electrophoresis: OD260/OD280=2.0,28S and18S was very clear. So, the concentration and the purity was good, meet the requirement of constructing cDNA library. The fragment of the purified ds cDNA was500-2500bp, it can be transformed to competent Y187yeast cells. The transformation efficiency of the cDNA library was2.5×107cfu/3ug PGADT7-Rec, the density was5×106cfu/mL, recombination fraction was100%, the average fragment of inserted cDNA was600bp. All the results indicated that the quality of the cDNA library was very good, it can be used for yeast two-hybrid. It also provided important materials and basement for screening cell receptor of GPV. The results can be used for researching the pathogenic mechanism and field planting regularity of GPV.