Abstract：
Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.
摘要：
Arazyme是一种细胞外金属蛋白酶，由称为蛋白沙雷氏菌的革兰氏阴性共生细菌分泌。对无花果的各种生物活性的研究有限。这项初步研究旨在研究重组阿曲霉素（rAra）的体外和体内抗癌和抗炎能力。ArazymeGene,使用pET-28a作为载体,在大肠杆菌BL21(DE3)中克隆和表达araA。使用镍柱纯化以获得纯的rAra。SDS-PAGE和蛋白质分析用于鉴定产物并测量蛋白质含量，分别。进行脱脂乳测试和酪蛋白测定以评估蛋白酶活性。将作为乳腺癌细胞模型的MCF7细胞暴露于不同浓度的rAra以使用MTT测定研究抗乳腺癌潜能。使用鼠气囊模型研究了rAra的抗炎特性。PCR和SDS-PAGE数据显示rAra的克隆和表达是成功的,并且在52KDa观察到感兴趣的酶。蛋白质测定表明通过纯化获得lmg/ml的rAra。脱脂乳琼脂上酶周围的清晰区域证实了rAra的蛋白水解活性，酪蛋白测定中的酶活性为320U/mg蛋白质。报告为IC50的rAra的细胞毒性作用在24小时和48小时后为16.2µg/ml和13.2mg/ml，分别。在气囊模型中，中性粒细胞计数和髓过氧化物酶活性，这是炎症的衡量标准，显着减少。结果表明，rAra可用于未来的机理研究和制药行业的研发活动中，以研究重组阿霉素的安全性和有效性。
