家蚕微孢子虫丝氨酸蛋白酶抑制剂蛋白serpin基因NbSPN106的鉴定

Identification of a new serpin gene (NbSPN106) from Nosema Bombycis

摘要:

摘要：【目的】Serpin在病原与宿主互作中起着重要的作用。本研究旨在分析在家蚕微孢子虫中的丝氨酸蛋白酶抑制剂蛋白（Serpin）的结构特征，原核克隆表达以及Western blotting检测。【方法】 基于家蚕微孢子虫全基因组序列，同源序列比对搜索获得serpin基因序列。利用在线软件分析基因的序列特征，ClustalX对氨基酸序列进行多重序列比对。构建含有GST标签的pGEX4T1-NbSPN106原核重组表达载体，在大肠杆菌BL21（DE3）诱导表达并进行纯化。纯化的重组蛋白免疫小鼠，制备抗体，并与家蚕微孢子虫总蛋白进行Western blotting免疫杂交。【结果】比对搜索在家蚕微孢子虫基因组中发现一个新的serpin基因NbSPN106。NbSPN106蛋白序列长度为384 aa，N端具有信号肽，编码一个42 kDa左右的成熟蛋白。多重序列比对说明NbSPN106具有保守的serpin位点，可能具有抑制功能。免疫杂交在家蚕微孢子虫总蛋白检测到一条45 kDa左右的特异条带。【讨论】生物信息学分析以及免疫杂交结果说明在家蚕微孢子虫中存在NbSPN106。这是在微孢子虫中首次报道发现serpin基因。对研究家蚕微孢子虫与宿主家蚕的互作具有一定的参考价值。

Abstract:

Abstract: [Objective] Serpins from pathogens have been implicated in evasion of the host immune system. We identified a new serpin protein (NbSPN106), analyzed its sequences, and detected using Western blotting. [Methods] Nosema bombycis proteins with an expect score less than 1×10-5 were checked against MEROPS database (http://merops.sanger.ac.uk) by Local Alignment Search Tool search and confirmed with Database of Protein Families and HMMS. Multiple sequence alignments were performed using the ClustalX. The sequence encoding the mature protein was amplified by PCR, cloned into the pGEX4T-1 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay. [Results] A new serpin gene, named NbSPN106, was identified form Nosema Bombycis genome. The coding sequence of this gene is 1155 bp length with a putative signal peptide and contains the conserved serpin sequences. A specific band of approximately 45 kDa was recognized by the anti-NbSPN106 serum. [Conclusions] The finding of serpins in Nosema bombycis raises new questions about their possible role in pathogenicity, which deserves further studies.