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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 study the physicochemical properties and biological functions of recombinant Acanthamocystis cv. Methods The prokaryotic expression plasmid pGEX-4T-1-AcCystatin was induced and purified. The purified AcCystatin was purified by enzyme digestion, and the recombinant protein was identified by Edman degradation N-terminal amino acid sequencing. The UV-vis spectra, fluorescence spectra, circular dichroism spectra and the inhibitory activities against human Cathepsin B, Cathepsin G, Cathepsin L and Cathepsin S were tested respectively. The physicochemical properties of AcCystatin were analyzed. Results The recombinant AcCystatin protein was purified and its molecular weight was about 13,600 after digestion. The purification result was good, and the result of amino acid sequencing was exactly the same as the theoretical amino acid sequence. The results of spectroscopy showed that disulfide bonds could be formed in the recombinant AcCystatin. The secondary structure of α-helix, β-sheet and random coil accounted for 39.57%, 35.28% and 25.25% respectively. Enzyme inhibition experiments showed that AcCystatin significantly inhibited Cathepsin B, Cathepsin L and Cathepsin S enzyme activity, but had no significant effect on CathepsinG enzyme activity. Conclusions The recombinant AcCystatin obtained in this study has high yield, high purity, normal folded protein structure and good biological activity, which lays an experimental foundation for the further study on the mechanism of AcCystatin immunoregulation.