准噶尔小胸鳖甲融合抗冻蛋白的免疫检测和抗冻活性分析  

Immunoassay and Antifreeze Detection Activity of Microdera punctipennis dzhungarica Fusion Antifreeze Protein Expressed in Escherichia coli

作　　者：薛娜[1] 赵干[1] 张富春[1] 马纪[1] 

机构地区：[1]新疆大学生命科学与技术学院分子生物学重点实验室,新疆生物资源基因工程重点实验室,乌鲁木齐830046

出　　处：《农业生物技术学报》2006年第6期865-869,共5页Journal of Agricultural Biotechnology

基　　金：科技部重大基础研究前期研究专项(No.2003CCA01000)资助。

摘　　要：利用克隆的新疆荒漠昆虫准噶尔小胸鳖甲(Microderapunctipennisdzhungarica)抗冻蛋白基因MpAFP5的全长序列设计PCR引物,构建原核表达质粒pMALp2x-MpAFP5,以麦芽糖融合蛋白(MBP)方式在大肠杆菌(Escherichiacoli)TB1中进行表达,经Amylose树脂亲和层析纯化获得了较高纯度的MBP-MpAFP5融合蛋白。Westernblot结果证明,MBP-MpAFP5融合蛋白在大肠杆菌中得到特异性的正确表达。细菌抗冻实验结果表明,导入MpAFP5基因的大肠杆菌经诱导表达在低温处理时抗冻能力无显著提高,而外加纯化的MBP-MpAFP5融合抗冻蛋白对细菌有明显保护作用,并随浓度升高而保护作用增强。Insect antifreeze protein gene MpAFP5 from Microdera punctipennis dzhungarica was cloned into pMALp2x vector and expressed in Escherichia coli （TB1） as a fusion form of Maltase-binding protein （MBP-MpAFP5） induced by IPTG. The fusion antifreeze protein was purified according to MBP-protein purification system.Western-blotting analysis showed that the MBP-MpAFP5 fusion protein was expressed successfully. The biological activity of this fusion antifreeze protein was measured by the relative survival rate of bacteria at low temperature. Results showed that the MpAFP5 expressed within bacteria no significant effect on the relative survival rate of bacteria, while the purified fusion protein added to bacteria culture medium could protect the bacteria from low temperature damage, and the protective effect was correlated with the antifreeze protein concentrations.

关 键 词：准噶尔小胸鳖甲 抗冻蛋白 原核表达 免疫检测 抗冻活性 

分 类 号：S188[农业科学—农业基础科学]