土耳其斯坦东毕吸虫成虫cDNA文库的构建

Construction of cDNA library for mature worm of Orientobilharzia turkestanicum

按照SMARTcDNA文库构建试剂盒说明 ,以TRIZOL试剂提取的土耳其斯坦东毕吸虫成虫总RNA为模板 ,以含SfiⅠB酶切位点的oligo(dT)为引物反转录合成第一链cDNA ,利用含SfiⅠA酶切位点的SMART核苷酸作为cDNA第一链在mRNA 5’端延伸出去的模板 ,采用LD -PCR技术以改良的oligo(dT)引物合成双链cDNA ,经蛋白酶K和SfiⅠ消化后 ,过CHROMASPIN - 4 0 0柱进行分级分离。双链cDNA与载体λTripEx2两臂连接 ,体外包装后构建成土耳其斯坦东毕吸虫成虫的cDNA噬菌体表达文库。经测定文库容量为 6 38× 10 6,文库扩增后的滴度为 3 4 8× 10 10 (Pfu/ml) ,PCR测定的重组率为 92 %。各项指标表明 ,我们成功的构建了高质量的cDNA文库 ,为进一步筛选、克隆保护性抗原基因奠定了基础。

Total RNA of mature worm of Orientobilharzia turkestanicum was extracted by Trizol and used to construct cDNA by library using the SMART cDNA by library construction kit.and the “anchor first-strand cDNA' containing asymmetrical Sfi Ⅰ restriction enzyme sites(A&B) was synthesized by transcription of total RNA.The double-strand cDNA 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 cDNA 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 cDNA phage expression library was constructed.The titer of un-amplified cDNA libraries was 6.38×106 and the titer of amplified cDNA libraries was 3.48×10 10(Pfu/ml).The percentage of recombinant clones of cDNA library was 92% by using PCR identification.These data show that a cDNA library with high quality has been successfully constructed and can be used to screen and clone the protective antigen genes.