洛氏脊漠甲抗冻蛋白基因的克隆、表达和功能检测

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。

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

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.