摘要
为了克隆和研究具有免疫原性的长角血蜱基因。应用建库试剂盒成功构建了长角血蜱雌蜱唾液腺cDNA表达文库．在无Rnase污染的环境下摘取半饱血长角血蜱雌蜱唾液腺。并从中提取RNA，进而纯化mRNA。利用oligo（dT）引物反转录合成双链cDNA，并在其两端加EcoRⅠ／HindⅢ定向接头并酶切，过柱分级分离后。将cDNA分子定向连接到具有EcoRⅠ／HindⅢ粘性末端的λSCREEN载体中．经体外包装转染E．coliERl647宿主菌，进行文库容量测定和扩增．以扩增文库的DNA为模板。利用载体通用引物检测cDNA文库中插入片断的大小和质量．经检测。所构建长角血蜱cDNA文库的基础库容量约为4．2×10^6pfu／mL，插入片段主要集中在300～2000bp之间．说明文库质量高、代表性强。
Total RNA and subsequently mRNA were isolated from salivary gland organs dissected from artially engorged Haemaphysalis longicornis ticks. A library of oligo（dT）-primed eDNA with added diectional EcoR Ⅰ/Hind Ⅲ linkers was constructed and ligated to the EcoR I/Hind III arms of the λ-creen vector. The recombinant phage DNA was packaged by using Phage Maker packaging extracts,resulng in a primary eDNA library with a size of 4.2 × 10^6 PFU. The library was proved to have good quality y all the control tests.
出处
《甘肃农业大学学报》
CAS
CSCD
2007年第6期13-17,共5页
Journal of Gansu Agricultural University
基金
国家基础平台项目(2005DKA21205-3)
国家高新技术研究发展计划(863)项目(2006AA10A207)
甘肃省自然基金项目(3ZS041-A25-035)
关键词
长角血蜱
CDNA表达文库
构建
Haemaphysalis longicornis
cDNA expression library
construction
