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绵羊痒螨(Psoroptes ovis)cDNA表达文库的构建

The construction of a cDNA expression library for the sheep scab mite Psoroptes ovis.

作者信息

Lee A J, Isaac R E, Coates D

机构信息

School of Biology, The University of Leeds, UK.

出版信息

Vet Parasitol. 1999 Jun 30;83(3-4):241-52. doi: 10.1016/s0304-4017(99)00061-8.

Abstract

The need for alternative control strategies for sheep scab is critical. One approach is to develop vaccines based on 'concealed' antigens derived from Psoroptes ovis. This strategy requires the identification and characterisation of potential target antigens, which has been hampered by the problem of limited biological material for isolation of protein antigens. To aid the discovery of P. ovis antigens and to provide a resource for generating recombinant protein, we constructed a P. ovis cDNA expression library, using total RNA isolated from 250 mg of mixed-stage P. ovis and the Clontech SMART cDNA synthesis kit. The presence of P. ovis-specific sequences was confirmed using PCR amplification and sequencing of actin. The sequences of cDNA inserts from six random clones included one with high homology to the Dermatophagoides pteronyssinus (house dust mite) antigen p Dp15. This is a glutathione S-transferase known to be an important house dust mite antigen. We conclude that this library will be a useful tool for the identification of potential target antigens for the immunological control of P. ovis and to further our understanding of the pathology of sheep scab.

摘要

迫切需要针对绵羊疥癣的替代控制策略。一种方法是基于源自绵羊痒螨的“隐蔽”抗原开发疫苗。该策略需要鉴定和表征潜在的靶抗原,但由于用于分离蛋白质抗原的生物材料有限这一问题而受到阻碍。为了帮助发现绵羊痒螨抗原并提供产生重组蛋白的资源,我们使用从250毫克混合阶段的绵羊痒螨中分离的总RNA和Clontech SMART cDNA合成试剂盒构建了一个绵羊痒螨cDNA表达文库。使用肌动蛋白的PCR扩增和测序确认了绵羊痒螨特异性序列的存在。来自六个随机克隆的cDNA插入片段的序列包括一个与粉尘螨抗原pDp15具有高度同源性的序列。这是一种已知为重要粉尘螨抗原的谷胱甘肽S-转移酶。我们得出结论,该文库将成为鉴定绵羊痒螨免疫控制潜在靶抗原以及进一步了解绵羊疥癣病理学的有用工具。

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