Construction of cDNA library for mature worm of Orientobilharzia turkestanicum

Total RNA of mature worm of Orientobilharzia turkestanicum was extracted by Trizol and used to construct by library using the SMART by library construction kit.and the “anchor first-strand cDNA containing asymmetrical Sfi Ⅰ restriction enzyme sites(AB) was synthesized by transcription of total RNA.The double-strand of full-length sequences was synthesized by LD-PCR by using a modified oligo(dT) primer and an anchor primer as the primer set,and anchor first-strand as the template.After digestion with Proteinase K and Sfi Ⅰ,the ds-cDNA was fractionated by CHROMA SPIN-400 Column.The optimal dscDNA were ligated into the Sfi Ⅰ-digested λTripEx2 vector.After package in vitro,the phage expression library was constructed.The titer of un-amplified libraries was 6.38×106 and the titer of amplified libraries was 3.48×10 10(Pfu/ml).The percentage of recombinant clones of library was 92% by using PCR identification.These data show that a library with high quality has been successfully constructed and can be used to screen and clone the protective antigen genes.