Construction of cDNA expression library from eggs of Haemaphysalis longicornis
2008
Liu Guangyuan | Tian Zhancheng | Cai Xuepeng
中国人. 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。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。所构建长角血蜱卵cDNA表达文库的基础库容量约为1.38×106 PFU，重组率为100%，扩增后文库的滴度为2×109 PFU•ml-1。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%。成功构建了长角血蜱卵cDNA表达文库。
显示更多 [+] 显示较少 [-]英语. cDNA expression library from eggs of Haemaphysalis longicornis was constructed for further screening and cloning of the potential candidate antigenic genes. 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. The size of the primary cDNA library was 1.38×106 PFU with titer of the amplified cDNA library of 2×109 PFU•ml-1. A full length cDNA encoding follistatin-related protein was cloned from the cDNA library by PCR. The cDNA expression library from eggs of H. longicornis was successfully constructed.
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