Construction and Characterization of a cDNA Library for Male Adult Ascaris suum

The objective of the present study was to construct a cDNA library for male adult of Ascaris suum with SMART(switching mechanism at 5′end of RNA transcript) technique using Creator~(TM) SMART~(TM) cDNA library construction kit.The total RNA was extracted from A.suum male adult using TriPure isolation reagent and mRNA was purified using Poly(A)Purist~(TM) kit.Single-strand cDNA was synthesized using PowerScript~(TM) reverse transcriptase,and then double-strand cDNA was synthesized and amplified by long-distance PCR(LD-PCR).The PCR products were digested by proteinase K and purified.After digestion with SfiⅠ and size fractionation(using) CHROMA SPIN-400TM columns,SMART cDNA was ligated to the Sfi I-digested,dephosphorylated pDNR-LIB vector.The ligation mixture was transformed into E.coli DH5α by electroporation.The constructed cDNA library contained 7.26×10~5 independent clones.The recombination rate was 96.7%.The average cDNA insert size was 1 kb.After the library was amplified,its capacity was 6.359×10~9 cfu/mL.A cDNA library for A.suum male adult was successfully constructed using SMART technology,which provides foundation for the screening and isolation of male-specific genes from A.suum.