亚洲璃眼蜱P27基因的克隆和表达模式分析

Molecular Cloning and Expression Patterns Analysis of the P27 Gene from Hyalomma asiaticum Ticks

为了进一步研究蜱吸血时唾液腺的基因表达调控机制,并为蜱疫苗的制备筛选功能性抗原,本试验对从亚洲璃眼蜱雌蜱吸血前后唾液腺消减文库中筛选出的一个具有Poly(A)尾的EST序列P27进行了研究。首先运用RACE技术扩增出该基因的5′端,在此基础上设计引物,扩增出该基因全长,此基因全长827 bp,其开放阅读框从第35个碱基始至第706个碱基止,预测分子量为27.28 ku,等电点4.22。生物信息学结构分析表明该基因含有跨膜区及多个活性位点,可能与细胞内DNA的转录相关;该基因与现有其他基因同源性很小,为一新基因;RT-PCR结果显示该基因在半饱血雌蜱的唾液腺、蜱壳、中肠均有表达,但在壳中表达丰度稍低。本研究克隆的P27基因序列已登录GenBank,序列号为EU180067。

In order to explore the regulation mechanism of the tick salivary glands by blood feeding, and screen vaccine candidates, a EST with poly（A） tail from the subtractive cDNA library of the hard tick-Hyalomma asiaticum was further studied. 5′rapid amplification of cDNA ENDS （RACE） were used to clone the full length cDNA. The full length of the gene named P27 is 827 bp and the coding region is from 35 bp to 706 bp. The putative protein of P27 is 27 kDa,and the isoelectric point is 4.22. The structure analysis showed that it contain a cross-membrane region and many activated loci, which suggest its transcript action in cell. Comparison of deduced amino acid sequences showed low homology with reported protein in the database. Expression analysis by RT-PCR revealed that P27 was expressed in salivary gland, shell and midgut of fed tick, but not in the unfed tick, which indicates P27 is induced to express by tick blood feeding.