重组广州管圆线虫半胱氨酸蛋白酶抑制剂(AcCystatin)的理化性质研究

Physico-chemical properties of recombinant AcCystatin, a cysteine protease inhibitor from Angiostrongylus cantonensis

目的研究重组广州管圆线虫半胱氨酸蛋白酶抑制剂(AcCystatin)的理化性质及其生物学功能。方法原核表达质粒pGEX-4T-1-AcCystatin经诱导表达、纯化酶切后获得纯化AcCystatin,采用Edman降解法N-端氨基酸测序进行重组蛋白鉴定。分别检测其紫外光谱、荧光光谱、圆二色谱和对人源Cathepsin B、Cathepsin G、Cathepsin L和Cathepsin S酶的抑制活性,分析AcCystatin理化性质。结果重组AcCystatin蛋白经纯化、酶切后相对分子质量约为13600,纯化效果良好,氨基酸测序结果与理论氨基酸序列完全相同。光谱学检测结果显示重组AcCystatin可形成二硫键,二级结构中α-螺旋、β-折叠和无规卷曲分别占39.57%、35.28%和25.25%。酶抑制实验结果显示AcCystatin可显著抑制Cathepsin B、Cathepsin L和Cathepsin S酶的活性,但对CathepsinG酶活性无明显作用。结论获得的可溶性AcCystatin重组蛋白产量大、纯度高、蛋白结构折叠正常,具有良好的生物学活性,为AcCystatin免疫调节机制的深入研究奠定了实验基础。

Objective To express, purify, identify and analyze the physico-chemical properties of recombinant AcCystatin. Methods Recombinant AcCystatin was separated and purified through GST-resin affinity chromatography and digestion of thrombin. The soluble purified protein was identified by SDS-PAGE and amino acid sequencing , followed by physico-chemical properties study on ultraviolet spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy of AcCystatin and its inhibitory activity against human Cathepsin B , G , L and S. Results The purified AcCystatin with a molecular weight of 13 600, was detected through SDS-PAGE and the first 20 amino acid residues were identified by N-terminal protein sequencing （Edman degradation method）. Anaylsis of AcCystatin ultraviolet spectrum, fluorescence spectrum and circular dichroism spectrum showed that AcCystatin contained a disulfide bond within the peptide chain , and the protein structure characterized by folding of the peptide chain into 3 types of secondary structures： alpha-helix （39.57%）, beta-sheet （35.28%） and random coil （25.25%） . Furthermore , AcCystatin was demonstrated to possessed an obvious inhibitory activity against human Cathepsin B（IC50=57.04 nmol/L）, L（IC50=89.28 nmol/L） and S（IC50=66.59 nmol/L）, but no significant effect on Cathepsin G activity. Conclusion Based on previous research, we have obtained recombinant AcCystatin in high production, high purity, high solubility and strong biological effect, which provides basic parameter for further study on mechanism of immunoregulation of AcCystatin in angiostrongyliasis cantonensis.