作　　者： ; ; ; ; ; ;

机构地区： 华南农业大学兽医学院

出　　处： 《畜牧兽医学报》 2006年第8期804-808,共5页

摘　　要： 采用Tripure Isolation Reagent抽提猪蛔虫雄虫成虫的总RNA,用poly(A)PuristTM纯化试剂盒分离mR-NA。分离mRNA后,用Clontech公司的CreatorTMSMARTTMcDNA文库构建试剂盒构建了猪蛔虫雄虫成虫的cDNA文库。结果获得了7.26×105独立克隆,重组率达96.7%,插入片段的平均长度约为1 kb。猪蛔虫雄虫cDNA文库的成功构建为利用文库筛选雄虫差异表达基因提供了材料来源,为研究猪蛔虫性别发育的分子机制奠定了基础。 The objective of the present study was to construct a cDNA library for male adult of Ascaris suurn 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. suurn 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 doublestrand cDNA was synthesized and amplified by long-distance PCR （LD-PCR）. The PCR products were digested by proteinase K and purified. After digestion with Sfi Ⅰ 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 eDNA insert size was 1 kb. After the library was amplified, its capacity was 6. 359×10^9 cfu/mL. A cDNA library for A. suurn male adult was successfully constructed using SMART technology, which provides foundation for the screening and isolation of male-specific genes from A. suum.

关 键 词： 猪蛔虫 雄虫 文库 技术 猪

领　　域： [农业科学] [农业科学] [农业科学] [生物学]