用SMART技术构建猪蛔虫雌虫cDNA文库

Construction of a cDNA library for female Ascaris suum using SMART technique

为克隆出与猪蛔虫产卵或发育相关基因的全长序列,本研究根据SMART(Switching mechanism at5'end of RNA transcript)技术,采用Clontech公司的CreatorTM SMARTTM cDNA Library Construction Kit构建了猪蛔虫雌虫的全长cDNA文库。用TriPure Isolation Reagent和Poly(A)PuristTM试剂盒提取mRNA后,以逆转录酶PowerScriptTM反转录合成第一链cDNA,然后通过LD-PCR合成并扩增ds cDNA。扩增产物经纯化、SfiⅠ酶切、过CHROMA SPIN-400TM柱去除小片段后,连接到SfiⅠ消化过的pDNR-LIB(带有SfiⅠA和B两个位点)质粒载体中,最后用电转化法将重组质粒转化到E.coli DH5α内得到原始文库,扩增后的文库保存在96孔细胞培养板中。经测定,构建的cDNA文库约含有1.6×106个重组子,重组效率达100%,插入片段多在0.4kb～2kb之间,平均插入片段长度约1.2kb,扩增后文库滴度为3×109CFU/mL。结果表明猪蛔虫雌虫的全长cDNA文库已构建成功,从而为筛选猪蛔虫雌虫的功能基因全长序列奠定了基础。

A cDNA library for female A.suum was constructed by SMART technique using Creator^TM SMART^TM cDNA Library Construction Kit. The total RNA was extracted from female A.suum using TriPure Isolation Reagent and mRNA was purified using Poly （A） Purist^TM kit. Single-strand eDNA was synthesized using PowerScript^TM reverse transcriptase, and double-strand eDNA was synthesized and amplified by long-distance PCR, The PCR products were digested by proteinase K and purified. After digestion with Sfi I and size fractionation using CHROMA SPIN-400^TM Columns, SMART eDNA was ligated to the Sfi I-digested, dephosphorylated pDNR-LIB vector. The ligation mixture was transformed into E.eoli DH5 ct by electroporation. The eDNA library contained 1.6 ×10^6 independent clones with DNA inserts of 0.4 kb-2 kb. The recombination rate was 100 %. This library could serve as a valuable resource for screening and isolating female-specific genes for A.suum.