镰形扇头蜱唾液腺cDNA文库的构建

Construction of cDNA Library from Salivary Gland of Rhipicephalus haemaphysaloides haemaphysaloides

构建了镰形扇头蜱唾液腺 c DNA表达文库。用 Trizol试剂提取镰形扇头蜱唾液腺总 RNA,经反转录合成 c DNA第 1链 ,应用 L D- PCR扩增方法 ,合成双链 c DNA。用 Sfi 内切酶修饰此双链 c DNA,使形成两端分别带有 Sfi A和Sfi B的粘末端。经 CHROMA SPIN- 4 0 0柱纯化 ,收集 4 0 0 bp以上的双链 c DNA片段 ,将其连接于带有 Sfi A和 Sfi B末端的λ Tripl Ex2噬菌体载体 ,经体外包装后 ,以 XL1 - Blue为受体菌构建 c DNA表达文库。经测定 ,库容量为7.0 8× 1 0 5,重组率为 95 .2 %。扩增后的文库滴度为 1 .4 5× 1 0 9pfu/ m L。随机挑取 2 0个克隆转化到质粒 p Tripl Ex2 ,经抽质粒、测序得到

Total RNA was extracted from salivary gland of Rhipicephalus haemaphysaloides haemaphysaloides by Trizol regent.The first strand cDNA was synthesized and the second strand was amplified by Long-Distance PCR.The Chroma Spin-400 was applied to purify cDNA fragments longer than 400 bp.The purified fragments were ligated with λTriplEx2,and after packaging in vitro,the cDNA phage expression library was constructed by infecting XL_1-Blue.It showed that the library contained 7.08×10~5 clones,approximately 95.2% of which were recombinant.The titer of the amplied library was 1.45×10~9 pfu/mL.20 clones that picked out from cDNA library at random were converted into plasmid pTriplEx2 and sequenced.Two full-length cDNA of cytochrome oxidase genes were obtained.