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小亚璃眼蜱4D8基因的克隆与原核表达
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赵金国1,2，曾巧英1，殷宏2，刘爱红2，李有全2 ，罗建勋2*
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(1.甘肃农业大学 动物医学院，甘肃 兰州730070；2.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，甘肃 兰州730046 )
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摘要：
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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。 |
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关键词：
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小亚璃眼蜱；4D8基因；克隆；原核表达 |
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中图分类号：S 852.746：Q 786 文献标识码：
A 文章编号：1673-4696(2008)06-0461-04 |
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Cloning and prokaryotic expression of 4D8 gene of
Hyalomma anatolicum anatolicum |
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ZHAO Jin-guo1,2， ZENG Qiao-ying1,YIN
Hong2， LIU Ai-hong2,LI You-quan2,LUO
Jian-xun2 |
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（1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China；2.Key Laboratory
of Animal Parasitology of Gansu Province/State Key Laboratory of Veterinary Etiological Biology/
Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China） |
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Abstract: 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.marginatum 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 coli.SDS-PAGE analysis showed that the expressed fusion protein was 45ku in molecular mass.The protein could be recognized by rabbit anti-Hyalomma anatolicum anatolicum salivary gland serum in Western-blotting. |
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Key words: Hyalomma anatolicum anatolicum;4D8 gene;cloning;prokaryotic expression
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