黑眶蟾蜍皮肤丝氨酸蛋白酶抑制剂基因的克隆与序列分析

目的]克隆黑眶蟾蜍皮肤丝氨酸蛋白酶抑制剂基因。方法]从海南海口产黑眶蟾蜍皮肤中抽提总RNA,经mRNA纯化后构建黑眶蟾蜍皮肤cDNA文库。根据已报道的丝氨酸蛋白酶抑制剂(Serpin)序列,设计寡核苷酸引物用于筛选、克隆、测序。结果]从所构建的cDNA文库中得到1个Serpin基因,命名为BmSP。对cDNA序列推导出的氨基酸序列分析表明,该BmSP蛋白属于Serpin家族中SERPIN B亚家族,与鼠鳞状细胞癌抗原2(SCCA2)同源。该蛋白C末端含有一个RCL环(Reactive center loop regions)和一段高度保守的五肽。结论]与SERPIN B亚家族中其他类型蛋白氨基酸序列比较结果表明,黑眶蟾蜍BmSP与SERPIN B亚家族中其他类型成员氨基酸序列相似度较高,具有高度的保守性,可能在其防御天敌捕食的过程中发挥重要作用。

Molecular Cloning and Comparison of Serpin from Bufo melanostictus Skin

Objective] To clone the Serpin gene from Bufo melanostictus skin.Method] Total RNA was extracted from the skin of Bufo melanostictus produced in Haikou,and then the cDNA library of Bufo melanostictus skin was constructed using purified mRNA from the total RNA.Based on the published cDNA sequences of Serpins,oligonucleotide primers were designed and used to screen the cDNA library.Result] A Serpin named BmSP was obtained from the cDNA library.As indicated by the analysis of the amino acid sequence deduced from the cDNA sequence,the BmSP protein belonged to the SERPIN B sub-family of Serpin family,showing homology with SCCA2.The C-terminus of the BmSP contained a RCL region and a highly-conserved penta peptide.Conclusion] Compared with other T amino acid sequences in SERPIN B sub-family,the amino acid sequences in BmSP and SERPIN B from Bufo melanostictus had high similarity with high conservation,they played important roles in defending agasint natural enemy predation.