腾冲嗜热厌氧杆菌tte0732基因的表达及生物信息学分析

摘要

腾冲嗜热厌氧杆菌tte0732(Galu) 基因编码的TTE0732是温度依赖性蛋白.为研究其在热适应中的作用,应用PCR技术克隆腾冲嗜热厌氧菌tte0732基因,构建原核表达载体pET-28a::tte0732并在大肠埃希菌BL21表达TTE0732;通过qRT-PCR分析tte0732基因在50、60、75和80 ℃的RNA表达量;应用生物信息学软件分析Galu在嗜热菌和常温菌中编码氨基酸的基本理化性质.成功构建了原核表达载体pET-28a::tte0732并在大肠埃希菌BL21中得到高效表达,TTE0732分子质量大小为35 ku,主要以可溶性形式存在;qRT-PCR显示tte0732 mRNA在75和80℃高表达;生物信息学分析得出tte0732基因完整的ORF全长909 bp,编码302个氨基酸,其中Ile(I)、Leu(L)含量高于常温菌,编码蛋白为酸性亲水性蛋白,等电点为5.22,含有18个潜在的磷酸化位点,不存在跨膜结构、信号肽和糖基化位点.预测其蛋白质二级空间结构以α-螺旋、无规则卷曲、β-折叠为主.腾冲嗜热厌氧杆菌TTE0732蛋白是一种亲水性蛋白,在原核系统能高效表达,本研究结果对嗜热蛋白质的热稳定性机制的研究具有一定的参考.%The TTE0732 encoding tte0732 (Galu) gene is a temperature dependent protein in Thermoanaerobacter tengcongensis. In order to study its role in heat accomodation, the tte0732 of T. tengcongensis and was cloned using PCR and constructed a prokaryotic expression vector pET-28a::tte0732 and expressed TTE0732 in E. coli BL21,then adopting qRT-PCR to analyze the tte0732 gene RNA expression at 50 ℃, 60 ℃, 75 ℃ and 80 ℃; and applying bioinformatic soft ware to analyze basic physio-chemical properties of Galu encoded amino acids in thermophilic and mesophilous bacteria. The results showed that the prokaryotic expression vector pET-28a::tte0732 was successfully constructed and efficiently expressed in E. coli BL21,the size of molecular weight of TTE0732 was 35 Ku,mainly ex-isted in soluble form;qRT-PCR showed high expression of tte0732 mRNA at 75℃ and 80℃;bioinformatics analysis got the full length ORF of tte0732 was 909 bp,encoding 302 amino acids,among them Ile(I),Leu(L) were higher than those in mesophilous,and its encoding protein was acidic hydrophilic,the isoelectric point was at 5.22,contai-ning 18 potential phosphorylation sites, no exist membrane acrossing structure, signal peptide, and glycosylation sites. It was predicted that the secondary space structures of the protein mainly were in α-helix, rulelessly crimpy, and β-folding. The TTE0732 protein of T. tengcongensis was a hydrophilic protein, which could be expressed effi-ciently in prokaryotic system,the results of this study possessed certain enlightenment to study thermo-stability mecha-nism of thermophilic proteins.