长角血蜱卵cdna表达文库的构建-中国农业科学.pdfVIP

中国农业科学 2008,41(7):2204-2208 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2008.07.045 长角血蜱卵cDNA表达文库的构建 刘光远，田占成，才学鹏，李知新，谢俊仁，王 路，张 林，龚真莉 （中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室，兰州 730046 ） 摘要：【目的】以长角血蜱卵为材料构建高质量的长角血蜱卵cDNA表达文库，为进一步筛选克隆目的基因奠 定基础。【方法】在无RNA酶污染的环境下从长角血蜱卵中提取RNA，进而纯化mRNA，采取oligo（dT）引物合成 双链 cDNA，定向克隆到λSCREEN 载体。用 PhageMaker extract 对其进行体外包装以形成完整的噬菌体，转染大 肠杆菌ER1647，测定库容量。并用构建的cDNA文库克隆已知的长角血蜱促卵泡激素基因。【结果】所构建长角血 蜱卵 cDNA 表达文库的基础库容量约为 1.38×106 9 -1 PFU，重组率为 100%，扩增后文库的滴度为 2×10 PFU·ml 。获 得了目的基因，其序列与GenBank中的长角血蜱促卵泡激素（DQ248886）的核苷酸序列的同源性为97.8%。【结论】 成功构建了长角血蜱卵cDNA 表达文库。 关键词：长角血蜱；卵；cDNA表达文库；构建 Construction of cDNA Expression Library from Eggs of Haemaphysalis longicornis LIU Guang-yuan, TIAN Zhan-cheng, CAI Xue-peng, LI Zhi-xin, XIE Jun-ren, WANG Lu, ZHANG Lin, GONG Zhen-li (Key Laboratory of Veterinary Parasitology of Gansu Province/State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046) Abstract: 【Objective 】 cDNA expression library from eggs of Haemaphysalis longicornis was constructed for further screening and cloning of the potential candidate antigenic genes. 【Method 】 Total RNA was isolated from eggs of H. longicornis, mRNA were purified, and cDNAs were synthesized and ligated to λSCREEN vector. The recombinant phage DNA was packaged by using Phagemark packaging extracts and then transfected to E. coli ER1647 to obtain the cDNA expression library. 【Result 】The size