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亚洲璃眼蜱差异表达新基因P18的克隆和鉴定
引用本文:王玉霞,周金林,周勇志,龚海燕,向飞宇,程天印,刘毅. 亚洲璃眼蜱差异表达新基因P18的克隆和鉴定[J]. 中国预防兽医学报, 2006, 28(4): 396-400
作者姓名:王玉霞  周金林  周勇志  龚海燕  向飞宇  程天印  刘毅
作者单位:1. 中国农业科学院上海家畜寄生虫研究所,农业部重点开放性实验室,上海,200232;湖南农业大学,动物科技学院,湖南,长沙,410128
2. 中国农业科学院上海家畜寄生虫研究所,农业部重点开放性实验室,上海,200232
3. 湖南农业大学,动物科技学院,湖南,长沙,410128
基金项目:国家自然科学基金;科技部科技基础条件平台建设计划
摘    要:为了进行抗蜱和蜱传病疫苗的研究,本实验对亚洲璃眼蜱雌蜱吸血前后唾液腺消减文库中获取的一个全长编码基因P18进行了研究。该基因全长519bp,共编码170个氨基酸,分子量为18.36Ku,等电点为4.28。BLAST分析表明,该基因预测的氨基酸与肩突硬蜱、篦子硬蜱唾液腺的抗凝血小肽有30%~40%低度同源性。将该基因亚克隆到pET-32a( )表达载体,转化BL21(DE3)宿主菌,经IPTG诱导,重组融合蛋白以可溶性形式高效表达。将可溶性重组蛋白免疫小鼠后获得的抗血清。经免疫印迹分析表明,该重组蛋白抗体可特异性的识别半饱雌蜱唾液腺中的天然蛋白抗原,而未吸血雌蜱唾液腺中则不显现特异条带。RT-PCR结果进一步证实,该基因在蜱吸血后的唾液腺中差异表达。

关 键 词:亚洲璃眼蜱  唾液腺  基因  差异表达
文章编号:1008-0589(2006)04-0396-05
收稿时间:2005-02-04
修稿时间:2005-02-04

Identification and molecular characterization of a new gene with expression variation from Hyalomma asiaticum ticks
WANG Yu-xia,ZHOU Jin-lin,ZHOU Yong-zhi,GONG Hai-yan,XIANG Fei-yu,CHENG Tian-yin,LIU Yi. Identification and molecular characterization of a new gene with expression variation from Hyalomma asiaticum ticks[J]. Chinese Journal of Preventive Veterinary Medicine, 2006, 28(4): 396-400
Authors:WANG Yu-xia  ZHOU Jin-lin  ZHOU Yong-zhi  GONG Hai-yan  XIANG Fei-yu  CHENG Tian-yin  LIU Yi
Affiliation:1 .Key Laboratory of Animal Parasitology, Shanghai Institute Parasitology, Chinese Academy of Agricultural Sciences, Ministry of Agriculture, Shanghai 200232, China; 2.College of Animal Science and Technology,Hunan Agricultural University, Changsha 410128,China
Abstract:s:To control tick and tick-borne illness,it is necessary to develop antitick vaccine research.We report here the isolation and characterization of a cDNA that encodes P18 from a substract cDNA of mRNA library of Hyalomma asiaticum,which constructed based on its salivary glands' genes expression variation in different biological process.The nucleotide sequence of the cDNA contains a 519-base-pair Open Reading Frame with a coding capacity of 18.36 Ku,and predicts that P18 contains 170 amino acids including a 14-amino acid signal sequence and isoelectric point is 4.28.The deduced amino acid sequence shows a 30 % to 40 % amino acid homology to small peptides of I.Scapularis and I.ricinus and low homology with other species.After cleaving the signal peptide the residua was subcloned into expressing vector PET32a( ),and expressed in E.coli BL21(DE3).The recombinant protein shows a signal band by SDS-PAGE,the size is about 34 Ku.Western-blotting analysis suggests P18 recombind with TRX protein antiserum can only recognize the native salivary gland antigen isolated from patially engorged male ticks,control with unegorged ticks.RT-PCR also reflects the same.
Keywords:Hyalomma asiaticum ticks  salivavy gland  gene  expressim variation
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