摘要
根据GenBank上登录的边缘革蜱4D8基因序列设计1对引物,采用PCR技术自半饱血小亚璃眼蜱雌性成蜱唾液腺cDNA表达文库中扩增获得了4D8基因;将其连入pMD18-T载体,构建重组克隆载体pMD18-Hyaa4D8。测序分析表明,克隆的小亚璃眼蜱4D8基因与边缘革蜱4D8基因的核苷酸序列的同源性为89.2%。将此片段定向亚克隆到原核表达载体pGEX-4T-1,构建重组原核表达载体pGEX-4T-1-Hyaa4D8,表达并纯化重组蛋白,通过免疫印迹试验鉴定该重组蛋白的生物学活性。结果显示,该重组蛋白是分子质量为45ku的融合蛋白,且表达产物能被半饱血小亚璃眼蜱雌性成蜱唾液腺抗原免疫兔血清识别。
One pair of specific primers were designed according to the sequence of 4D8 gene of Dermacentor marginatum available in GenBank. The 4D8 gene was amplified by PCR from cDNA expression library of the salivary gland of semi-engorged female adult Hyalomma anatolicum anatolicum and cloned into pMD18-T vector to construct recombinant clonal vector pMD18-Hyaa4D8. Sequencing result showed that the nucleotide sequence of the cloned 4D8 gene shared 89.2% identity with that of D. rnarginatum published in GenBank. A recombinant expression plasmid pGEX-4T-1-Hyaa4D8 was constructed by subcloning the cloned 4D8 gene into the linearized pGEX-4T-1 vector. Then the plasmid pGEX-4T-1-Hyaa4D8 was expressed in Escherichia coll. SDS-PAGE analysis showed that the expressed fusion protein was 45 ku in molecular mass. The protein could be recognized by rabbit anti-Hyalomma anatolicum anatolicum salivary gland serum in Western-blotting.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第6期461-464,共4页
Chinese Veterinary Science
基金
国家自然科技资源共享平台项目(2005DKA21100)
国家高技术研究发展计划(863)项目(2006AA10A207)
国家“十一五”科技支撑计划项目(2007BAD40B06)
关键词
小亚璃眼蜱
4D8基因
克隆
原核表达
Hyalomma anatolicum anatolicum
4D8 gene
cloning
prokaryotic expression
