文章摘要
腾冲嗜热厌氧杆菌Cas2原核表达及生物信息学分析
Prokaryotic Expression and Bioinformatics Analysis of Cas2 in Thermoanaerobacter tengcongensis
  
DOI:doi:10.3969/j.issn.1005-7021.2021.02.002
中文关键词: 腾冲嗜热厌氧菌  cas2基因  qRT-PCR  原核表达  生物信息学分析
英文关键词: Thermoanaerobacter tengcongensis  cas2 gene  qRT-PCR  prokaryotic expression  bioinformatics analysis
基金项目:国家自然科学基金项目(31500067);甘肃省科技计划项目重点研发计划项目(18YF1NA077);甘肃农业大学2018年学生科研训练计划项目 (SRTP,20180327 20180328);甘肃省科学院应用研究与开发项目(2018JK-07,2019HZ-03,2020JK-03)
作者单位
王川 甘肃农业大学 动物医学院甘肃 兰州 730070 
刘原子 陕西梅里众诚动物保健有限公司陕西 西安 712034 
魏亚琴 甘肃省微生物资源开发利用重点实验室
甘肃省科学院生物研究所厌氧微生物中心甘肃 兰州 730000 
毛婷 甘肃省微生物资源开发利用重点实验室
甘肃省科学院生物研究所厌氧微生物中心甘肃 兰州 730000 
杨宇泽 北京市畜牧总站, 北京 100101 
俞海山 甘肃农业大学 动物医学院甘肃 兰州 730070 
张钊 甘肃农业大学 动物医学院甘肃 兰州 730070 
万学瑞 甘肃农业大学 动物医学院甘肃 兰州 730070 
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中文摘要:
      为研究CRISPR/Cas系统及其相关蛋白Cas2(TTE2657)在腾冲嗜热厌氧杆菌热适应中的作用,应用PCR技术构建了原核重组质粒pET-28a::cas2,并在大肠埃希菌BL21表达Cas2蛋白;结合生物信息学软件对cas2编码蛋白的基本理化性质、氨基酸同源性、空间结构及蛋白质相互作用网络进行预测和分析。结果显示,成功构建了原核表达载体pET-28a::cas2并在大肠埃希菌BL21中得到表达,Cas2分子质量大小为9.9 ku,主要以可溶性形式存在;qRT-PCR显示cas2 mRNA在60 ℃和75 ℃高表达;生物信息学分析显示cas2基因其完整的ORF全长264 bp,编码88个氨基酸,其中Ile(14)、Ser(14)、Phe (12)含量较高,等电点为9.31,不存在跨膜结构。其蛋白质二级空间结构以α-螺旋、无规则卷曲、β-折叠为主,蛋白互作预测网络显示Cas2与Cas3、Cas5、Cas7等其家族大部分蛋白存在相互作用。进化树分析显示腾冲嗜热厌氧杆菌cas2基因与厌氧菌芽胞杆菌B7M1同源性最高(39.5%)。腾冲嗜热厌氧杆菌cas2编码蛋白是一种亲水性蛋白,在原核系统能高效表达。本研究为嗜热蛋白质的热稳定性机制的研究提供参考。
英文摘要:
      In order to study the role of CRISPR/Cas system and its related protein Cas2 (TTE2657) in thermal adaptation of Thermoanaerobacter tengcongensis, a prokaryotic recombinant plasmid pET-28a:cas2 was constructed and Cas2 protein was expressed in E. coli BL21.Cas2 was used to predict and analyze the basic physicochemical properties, amino acid homology, spatial structure and protein interaction network combined with bioinformatics software. The results showed that the prokaryotic expression vector pET-28a:: cas2 was successfully constructed and Cas2 was expressed in E. coli BL21. The molecular mass of Cas2 was 9.9 ku, mainly in soluble form. qRT-PCR showed that cas2 mRNA was highly expressed at 60 ℃ and 75 ℃; and bioinformatics analysis showed that the complete ORF of cas2 was 264 bp in length and encoded 88 amino acids. Among them the contents of Ile(14), Ser(14) and Phe(12) were higher, and the isoelectric point was at 9.31, and there was no transmembrane structure. The secondary spatial structure of the protein was mainly α-helix, irregular coil, and β-folded. The protein interaction prediction network showed that Cas2 interacted with the most of the family proteins Cas3, Cas5, Cas7, and others. Phylogenetic tree analysis showed that the gene cas2 of T. tengcongensis had the highest homology with Bacillus anaerobic B7M1. And the cas2 coded protein of T. tengcongensis was a hydrophilic protein that could efficiently express in prokaryotic systems. This study has some instructive for the study on thermal stability mechanism of thermophilic proteins.
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