长角血蜱卵cDNA表达文库的构建

 【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 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%。【结论】成功构建了长角血蜱卵cDNA表达文库。

Construction of cDNA Expression Library from Eggs of Haemaphysalis Iongicomis

Abstract: 【OBJECTIVE】The cDNA Expression Library, based on the source of eggs of Haemaphysalis longicornis, was constructed successfully for further screening the potential candidate antigens angist the Haemaphysalis longicornis. 【METHOD】Total RNA were isolated from eggs of Haemaphysalis Longicornis, subsequently mRNA were purified a library of oligo(dT)-primed cDNA with added directional EcoRⅠ/Hind Ⅲ linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoRⅠ/Eind Ⅲ arms of the λSCREEN vector. 【RESULT】The recombinant phage DNA was packaged by using Phagemarker packaging extracts, resulting in a primary cDNA library with a size of 1.38×106 PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2×109 PFU. A cDNA encoding follistatin-related protein was cloned from the expression library. 【CONCLUSION】The results suggested that the cDNA expression library was constructed successfully.