亚洲璃眼蜱差异表达新基因P18的克隆和鉴定

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。

Identification and molecular characterization of a new gene with expression variation from Hyalomma asiaticum ticks

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.