长角血蜱新基因HL05全长序列的克隆与原核表达  

Cloning and prokaryotic expression of a novel gene HL05 of Haemaphysalis longicornis

作　　者：张丽艳[1] 刘光远[1] 田占成[1] 谢俊仁[1] 龚真莉[1] 刘建刚[1] 

机构地区：[1]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农业部草食动物疫病重点开放实验室,甘肃兰州730046

出　　处：《中国兽医科学》2011年第4期380-386,共7页Chinese Veterinary Science

基　　金：国家高技术研究发展计划(863)项目(2006AA10A207);国家基础平台项目(2005DKA21205-3)

摘　　要：以兔抗长角血蜱饥饿雌蜱全蜱蛋白阳性血清为探针,对长角血蜱饥饿雌蜱cDNA表达文库进行免疫学筛选,获得一个阳性克隆HL05。经5′-RACE技术得到HL05基因的全长序列。HL05基因全长1 053bp,开放阅读框长540bp,编码179个氨基酸,预计蛋白分子质量为20.01ku。序列同源性分析表明,该基因为长角血蜱新基因。对推导的氨基酸序列进行生物信息学分析,表明HL05蛋白为分泌型蛋白,无跨膜区,具有较高的抗原指数。将该基因亚克隆后连接到原核表达载体pGEX-4T-1上,转入BL21(DE3)宿主菌,经IPTG诱导后得到大小为46ku左右的重组蛋白,与预期大小一致。Western-blot结果显示兔抗长角血蜱成蜱全虫抗体能够识别该重组蛋白。One positive clone of HL05 was obtained by immunoscreening from a cDNA library of Haemaphysalis longicornis unfed female tick with rabbit anti-H.longicornis serum.The full sequence of the gene was amplified by 5′ rapid amplification of cDNA ends protocol.HL05 gene contained an open rea-ding frame of 540 bp encoding a 20.01 ku protein of 179 amino acid residues.Sequence similarity analysis indicated that HL05 gene was a novel gene of H.longicornis.Bioinformatics analysis indicated that the protein was classified into secernent type without transmembrane domain,but with more potential antigenic determinants.After being subcloned into expression vector pGEX4T-1,it was expressed in Escherichia coli BL21（DE3）.The expressed recombinant protein was about 46 ku in size.Western-blot analysis indicated that the recombinant protein was able to recognize the antibody against H.longicornis.

关 键 词：长角血蜱 HL05新基因 全长 序列分析 原核表达 

分 类 号：S852.746[农业科学—基础兽医学]