高原链带藻抗氧化蛋白分离纯化与结构鉴定

Purification and structure determination of an antioxidant protein from plateau microalga Desmodesmus sp.

摘要:

【背景】微藻Desmodesmus sp. QL96从我国西藏地区分离得到，经形态鉴定隶属于链带藻属。前期研究发现，这种链带藻在4 °C和25 °C下均可生长，在25 °C生长时，干细胞中蛋白质含量可高达71.68% (质量分数)，而且蛋白粗提物具有一定的抗氧化活力。【目的】分离纯化Desmodesmus sp. QL96细胞中具有抗氧化活力的蛋白质，并对其结构进行鉴定。【方法】应用柱层析的方法分离纯化Desmodesmus sp. QL96细胞中具有抗氧化活力的蛋白质，通过化学发光法和细胞学实验对该蛋白的抗氧化活性进行检测，并通过质谱技术对其一级结构进行检测。【结果】Desmodesmus sp. QL96细胞中抗氧化蛋白的含量占微藻细胞干重的11.40% (质量分数)；纯化的Desmodesmus sp. QL96抗氧化蛋白在一定浓度范围内对OH?、DPPH、ABTS自由基和H2O2具有较好的清除率(超过60%)，细胞学实验显示其对H2O2诱导的HepG2细胞氧化损伤具有抑制作用，验证了其抗氧化功能；通过质谱技术检测了Desmodesmus sp. QL96抗氧化蛋白的氨基酸序列，并进行了生物信息学分析，结果显示，这种蛋白质的理论分子量为44.8 kD、pI 5.79，与NCBI中目前已知的其他物种蛋白质的相似性不超过59%。【结论】Desmodesmus sp. QL96可能生产一种具有抗氧化活性的新蛋白质，后续将对其转录本进行分析，验证其遗传信息的同源性，并分析其规模化生产和应用前景。

关键词:西藏高原，链带藻，抗氧化蛋白，蛋白纯化，蛋白结构

Abstract:

[Background] Microalga Desmodesmus sp. QL96 was isolated from Tibet Plateau, China. We had previously found that the microalga was able to grow at both 4 °C and 25 °C, and produced 71.68% (W/W) cellular proteins which showed antioxidant activities. [Objective] Herein, an antioxidant protein in Desmodesmus sp. QL96 cells was purified and its structure was analyzed. [Methods] Purification of the antioxidant protein was performed through chromatography. The antioxidant activity of the purified protein was determined using chemiluminescence assays and in vitro cell cultures. The structure of the purified protein was then analyzed using mass spectrometery. [Results] The antioxidant protein took up 11.40% (W/W) of the dry microalgal cells. The purified protein demonstrated scavenging capacity to free radicals of OH?, DPPH, ABTS and H2O2 (over 60% scavenged). In vitro cell cultures using liver carcinoma cell HepG2 demonstrated that the Desmodesmus sp. QL96 antioxidant protein could reduce oxidative damage to the cells. The amino acid sequence of the protein determined using a mass spectrometery showed that the molecular weight of the antioxidant protein was 44.8 kD with a pI of 5.79. The homologous rate of the protein with the known proteins in NCBI was below 59%. [Conclusion] Microalga Desmodesmus sp. QL96 produces a previously uncharacterized protein which demonstrates antioxidant activity. The transcript of the protein will be analyzed to verify the genetic sequence similarity in the future, as well as its potential function and industry applications.

Key words:Tibet Plateau, Desmodesmus sp., antioxidant protein, protein purification, protein structure