荒漠甲虫小胸鳖甲抗冻蛋白的酵母表达及应用

Yeast expression and application of an antifreeze protein from the desert beetle Microdera punctipennis

摘要:

昆虫抗冻蛋白 (Antifreeze protein，AFP) 的抗冻活性很高，可应用于生物组织和细胞的低温保存。为了在酵母中表达荒漠甲虫小胸鳖甲Microdera punctipennis抗冻蛋白MpAFP698，并确定其在低温下的保护作用，本文通过构建真核表达载体pPIC9K-Mpafp698，转化巴斯德毕赤酵母GS115，诱导表达小胸鳖甲抗冻蛋白MpAFP698。利用免疫印迹 (Western blotting) 分析MpAFP698蛋白的特异性表达，结果显示Mpafp698 基因可整合到酵母基因组中并分泌表达，且酵母自身蛋白很少分泌表达。检测抗冻蛋白的低温保护作用，结果发现，小胸鳖甲抗冻蛋白可显著改善冷冻小鼠肝脏等器官的细胞形态，降低血细胞在4 ℃的溶血率，提高SF9细胞冻融后的存活率。本研究表明，小胸鳖甲AFP可以在毕赤酵母中分泌表达，便于纯化，有良好的低温保护效果。

Abstract:

Insect antifreeze protein (AFP) has high antifreeze activity. Antifreeze proteins can be used in cryopreservation of biological tissues and cells. We expressed an antifreeze protein from the desert beetle Microdera punctipennis in yeast and determined the function of the protein at low temperatures. Yeast expression vector, pPIC9K-Mpafp698, was constructed and transformed into Pichia pastoris GS115. The expression of MpAFP698 was induced by methanol, and identified by tricine SDS-PAGE and Western blotting. Mpafp698 gene was inserted into the genome of the host yeast strain GS115, and correctly expressed. Hardly any yeast’s own protein was secreted into the media. Cryoprotective experiments showed that MpAFP698 can significantly protect mouse liver as well as other mouse organs from cold damage compared with those in the control of Bovine serum albumin (BSA) addition. Besides, the hemolysis of blood cells protected by MpAFP698 at 4 °C was reduced and the survival rate of SF9 cells protected by MpAFP698 after freezing and thawing was increased compared to those of the control with BSA addition. Our results showed that MpAFP698 can be expressed in yeast, which allows a convenient purification of the MpAFP protein that has the cryoprotective effect.