[关键词]
[摘要]
目的 克隆获取冬虫夏草指标蛋白氰酸酶(IP4)的cDNA全长及对其抗原表位预测。方法 结合课题组前期采用蛋白组学法筛选得到的冬虫夏草指标性蛋白IP4和冬虫夏草转录组数据,挖掘IP4 mRNA序列,并克隆IP4 cDNA全长,通过NCBI结构分析服务系统对IP4进行保守区和功能区分析,利用DNAMAN 6.0和DNAStar软件分别进行序列比对和抗原表位分析。结果 从转录组数据中挖掘到IP4的2个可变剪接体,属于内含子保留的可变剪接。RT-PCR结果显示,IP4 cDNA全长465 bp,编码154 aa(氨基酸)。IP4蛋白的保守区和主要催化活性位点位于87~154 aa,该区域为氰酸酶家族保守核心区域和二聚体结合区域,DNAMAN 6.0分析结果表明1~39 aa和55~81 aa区具有较高种属特异性,DNAStar预测IP4蛋白的抗原表位区主要分布于25~90 aa。结论 最终确定IP4的25~39 aa和55~81 aa为最佳抗原特异区,为后续规模化制备IP4蛋白特异抗原区及免疫法鉴定冬虫夏草奠定基础。
[Key word]
[Abstract]
Objective To clone the full-length of cDNA protein marker cyanase (IP4) of Ophiocordyceps sinensis and predict the antigenic sites. Methods The information of protein marker of O. sinensis was obtained by proteomics technology. The transcriptome database of O. sinensis and mycelium constructed in our lab were used to analyze IP4 unigenes and appropriate primers designed to amplify IP4, which was then cloned and sequenced. The conserved sequence of IP4, homology comparison, and the antigenic site were analyzed by DNAMAN6.0 and DNAStar software. Results Two alternative splice variants of IP4 were discovered from the transcriptome data and belonged to the invariant alternative splicing. The protein marker of O. sinensis was identified as cyanate hydratase, 465 bp, encoding 154 aa. The analysis of the conserved and functional regions showed that catalytic site and binding site mainly lied in C-terminal of IP4. Analysis of DNAMAN 6.0 showed that species 1-39 aa and 55-81 aa were of higher species specificity, and DNAStar results showed that the epitope region of IP4 protein is distributed from 25 to 90 aa. Conclusion The optimal antigen region of IP4 lied in the region 25-39 and 55-81 aa, which lays a foundation for the subsequent large-scale preparation of IP4 protein and eutilizing ELISA to identify authenticity of O. sinensis.
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[基金项目]
国家自然科学基金资助项目(30801522,81373920);四川省中医药管理局川产道地药材大品种项目;成都市科技局科技惠民项目资助