准噶尔小胸鳖甲融合抗冻蛋白活性的比较研究

为了比较准噶尔小胸鳖甲Microdera punctipennis dzhungarica原核表达的不同融合抗冻蛋白活性是否存在差异,分别利用重组表达质粒pGEX-4T-1-Mpafp5和pMAL-p2x-Mpafp5在大肠杆菌Escherichia coli BL21(DE3)中诱导表达融合抗冻蛋白GST-MpAFP5和MBP-MpAFP5,并利用亲和层析纯化蛋白。SDS-PAGE分析结果证明获得了两种高效表达纯化的GST-MpAFP5和MBP-MpAFP5融合蛋白。在浓度为0.5 mg/mL时,两种蛋白的热滞值分别为0.51℃和1.336℃,其溶液冰晶形态均为棱型。抗冻保护实验结果显示： 准噶尔小胸鳖甲两种融合抗冻蛋白均能够显著提高细菌在低温下的存活率,并且MBP-MpAFP5对细菌的保护效果显著高于GST-MpAFP5。研究结果对于准噶尔小胸鳖甲抗冻蛋白的应用具有重要的理论意义。

Comparison of activities of fusion antifreeze proteins from Microdera punctipennis dzhungarica (Coleoptera:Tenebrionidae)

In order to compare the activity differences between the prokaryotic expressed MBP-MpAFP5 and GST-MpAFP5, the two recombinant prokaryotic expression plasmids pGEX-4T-1-Mpafp5 and pMAL-p2x-Mpafp5 were expressed in Escherichia coli BL21(DE3) to obtain two different fusion antifreeze proteins of Microdera punctipennis dzhungarica. The two fusion proteins were purified by affinity chromatograph. SDS-PAGE analysis indicated that both of the fusion antifreeze proteins were highly expressed in soluble fractions. The thermal hysteresis activities （THA） of GST-MpAFP5 and MBP-MpAFP5 at a concentration of 0.5 mg/mL were measured as 0.51℃ and 1.336℃, respectively. The ice crystal morphology of both proteins displayed pyramid shape. Cryoprotection to bacteria experiments showed that the two fusion proteins could protect bacteria survival at subzero 7℃ temperature. Comparatively, MBP-MpAFP5 had higher protective activities than GST-MpAFP5. These results provide basic data for application of the insect antifreeze proteins in the future.