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

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.