›› 2008, Vol. 51 ›› Issue (5): 480-485.

• 研究论文 • 上一篇    下一篇


马 纪,王 芸,刘忠渊,张富春   

  • 出版日期:2010-07-27 发布日期:2008-05-20
  • 通讯作者: 张富春

Cloning and activity analysis of insect antifreeze protein gene from Pterocom a loczyi(Coleoptera: Tenebrionidae)

MA Ji   

  • Online:2010-07-27 Published:2008-05-20


为了研究抗冻蛋白基因是否在新疆荒漠昆虫洛氏脊漠甲 Pterocoma loczyi 中存在,利用 RT-PCR 技术克隆获得了洛氏脊漠甲抗冻蛋白的 cDNA 片段,命名为 Plafp743。测序结果表明洛氏脊漠甲 Plafp743 所编码的蛋白质由 94 个氨基酸组成,蛋白序列呈现规则结构 CTX1X2X3X4CX5X6X7X8X 9。利用原核表达载体构建重组质粒 pGEX-4T-1-Plafp743,转化 Escherichia coli BL21 进行融合蛋白表达,SDS-PAGE 结果表明抗冻蛋白PLAFP基因以可溶性融合蛋白形式表达,相对分子质量 36 kD。用 Glutathione Sepharose 4B 亲和柱对表达蛋白进行纯化后,通过赤翅甲 Dendroides canadensis 抗冻蛋白的小鼠抗血清进行 Western blot 分析,结果表明纯化的 GST-PLAFP 融合蛋白与赤翅甲抗血清能够发生特异性的免疫反应。大肠杆菌的低温抗冻保护实验结果证明,30 μg/mL 的 GST-PLAFP 在-6℃ 对细菌具有显著的保护作用,且随着抗冻蛋白浓度的增加,抗冻保护作用的效 果也随之增加。本研究首次报道了从荒漠昆虫洛氏脊漠甲扩增得到抗冻蛋白基因,提示抗冻蛋白可能是荒漠昆虫普遍采取的过冬生存策略,为进一步开发利用昆虫抗冻蛋白奠定了基础。

关键词: 洛氏脊漠甲, 抗冻蛋白, 克隆, 序列分析, 原核表达, 抗冻保护


To investigate antifreeze protein gene in the beetles liv ing in Xinjiang Junggar desert, Pterocoma loczyi, a
large size desert beetle, was chosen for antifreeze gene cloning. RT-PCR technique was employed to obtain Pterocoma loczyi antifreeze protein gene. One of the obtained cDNA fragment was named Plafp 743.Sequence analysis showed that Plafp 743 encodes 112 amino acids with the repeated constructive sequence CTX1X2X3X4CX5X6X7X8X9, which is the special motif for some of the known insects antifreeze proteins. Recomb inant plasmid pGEX-4T-1-Plafp743 was transferred into Escherichia coli BL21 and induced by IPTG. The expressive product was analyzed by SDS-PAGE, and the result showed that pGEX-4T-1-Plafp743 was expressed in fusion protein with relative molecul ar mass of 36 kD. The fusion protein, named GST-PLAFP, was purified by Glutathione Sepharose 4B, and then identified by Western blotting with mouse antiserum against antifreeze protein from Dendroides canadensis. Then, GST-PLAFP with different concentrations was added to E. coli culture for various days at -6℃ to check its cryoprotective effect on bacteria survival at subzero temperatures. This cryoprotective experiment showed that the fusion protein GST-PLAFP could protect bacteria from freezing to death at conce ntrations as low as 30 μg/mL. The results suggest that AFP might be a widely adopted strategy for overwintering of desert beetles.

Key words: Pterocoma loczyi, antifreeze p rotein (AFP), cloning, sequence analysis, prokaryotic expression, cryoprotecti ve effect