腾冲嗜热厌氧杆菌ahpC编码基因的克隆表达及生物信息学分析  

Cloning, expression and bioinformatics analysis of ahpC gene of Thermoanaerobacter tengcongensis

作　　者：郑航辉 高昇 杨宇泽 吴润[1] 万学瑞[1] 王川[1] 刘磊[1] ZHENG Hang-hui;GAO Sheng;YANG Yu-ze;WU Run;WAN Xue-rui;WANG Chuan;LIU Lei(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070;Beijing General Station of Animal Husbandry Service,Beijing 100101,China)

机构地区：[1]甘肃农业大学动物医学学院,兰州730070 [2]北京市畜牧总站,北京100101

出　　处：《生物学杂志》2019年第5期36-40,共5页Journal of Biology

基　　金：国家自然科学基金(31500067,31560700);兰州市科技创新项目(No.2014-2-11)

摘　　要：为了研究腾冲嗜热厌氧杆菌( Thermoanaerobacter tengcongensis )AhpC在嗜热中的作用,应用PCR技术扩增腾冲嗜热厌氧菌 tte0270 基因(即 ahpC 编码基因),构建了原核表达载体pET-28a:: ahpC 并在大肠杆菌BL21(DE3)表达 AhpC ,并通过荧光定量PCR分析 ahpC 在50 ℃、60 ℃、75 ℃和80 ℃的mRNA表达量;同时利用生物信息学软件分析AhpC在腾冲嗜热厌氧杆菌、单核细胞增生李斯特菌和大肠杆菌中编码氨基酸的基本理化性质和蛋白质三级结构。在BL21(DE3)中腾冲嗜热厌氧杆菌AhpC蛋白能高效的表达,以可溶性形式存在,分子质量为26 ku;荧光定量PCR结果表明腾冲嗜热厌氧杆菌 ahpC mRNA表达量随温度升高而提高;生物信息学分析得出AhpC含有220个氨基酸,为酸性亲水性蛋白,等电点为6.126,蛋白质有29.1%的氨基酸参与α-螺旋结构的形成。嗜热菌AhpC中Glu(E)和Lys(K)的含量高于常温细菌。腾冲嗜热厌氧杆菌AhpC的三级结构明显比大肠杆菌和单核细胞增生李斯特菌的更紧凑。结果对腾冲嗜热菌嗜热机制研究具有一定的启发。In order to study the role of AhpC of Thermoanaerobacter tengcongensis in thermophilic adaptation, the prokaryotic expression vector pET-28a :: ahpC was constructed and expressed in Escherichia coli BL21(DE3) using PCR to amplify the ahpC of T. tengcongensis . mRNA expression levels of the ahpC at 50 ℃, 60 ℃, 75 ℃ and 80 ℃ were analyzed by quantitative real time polymerase chain reaction (qRT-PCR). The mass of AhpC was 26 ku, which was mainly in soluble form. qRT-PCR analysis shown that the mRNA expression of ahpC was increased with the increase of temperature. Bioinformatics analysis showed that the ahpC gene was 663 bp in length and encoded 220 amino acids. The contents of Glu (E) and Lys (K) were higher than those of normal bacteria. It is predicted that 29.1% of the amino acids in the secondary structure of the protein are involved in the formation of α-helix structure. The tertiary structure of the AhpC in Thermoanaerobacter tengcongensis is significantly more compact than those of E. coli and Listeria monocytogenes . The results of this study have a certain help for the study of thermophilic adaptation of Thermoanaerobacter tengcongensis.

关 键 词：腾冲嗜热厌氧菌 AHPC 热适应 

分 类 号：Q78[生物学—分子生物学]