Jafari Zohreh, Bandehpour Mojgan, Gheflat Shivasadat, Mohammadi Nasrin, Kazemi Bahram
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Iran J Pharm Res. 2022 Jan 16;21(1):e123791. doi: 10.5812/ijpr.123791. eCollection 2022 Dec.
Ecarin is a metalloproteinase found in snake venom (SVMP) with an important role in coagulation and control of hemostasis. It can specifically produce active-thrombin from prethrombin-2 and does not differentiate between normal and abnormal prothrombin. It is used in diagnostic tests and to evaluate the treatment process of many diseases. There are many drawbacks associated with separating these compounds from snake venom. Therefore, in this study, full-length recombinant () was cloned, expressed, and purified in eukaryotic host cells. To determine the most effective form of the enzyme, r-Ecarin was compared with the recombinant truncated form, which has only the metalloprotease domain of the protein (r-Ecamet) in terms of function and expression. Briefly, A DNA construct composed of sequence-encoding was designed and cloned into pCAGGS expression vector and, subsequently, expressed in Chinese Hamster Ovary (CHO) cells. To identify the enzymatic activity of expressed protein, a bioactivity assay was performed. Blood coagulation time and expression levels of r-Ecarin and r-Ecamet proteins were compared. Also, a histopathological assessment was carried out on the liver of mice treated with these proteins. Comparison of r-Ecarin and r-Ecamet expression pattern demonstrated that full-length Ecarin expression has at least 2-fold higher expression in eukaryotic cells. Determination of r-Ecarin function proved that this protein is capable of prothrombin cleavage and producing thrombin. Comparison of PT test results between the r-Ecarin and r-Ecamet showed that there is a significant difference in the activity of the two enzymes and the full-length protein coagulates the blood in less time.
伊卡林是一种存在于蛇毒中的金属蛋白酶（蛇毒金属蛋白酶），在凝血和止血控制中起重要作用。它能从凝血酶原-2特异性产生活性凝血酶，且对正常和异常凝血酶原不做区分。它用于诊断测试以及评估多种疾病的治疗过程。从蛇毒中分离这些化合物存在许多缺点。因此，在本研究中，全长重组伊卡林在真核宿主细胞中进行了克隆、表达和纯化。为了确定该酶最有效的形式，将重组伊卡林与仅具有该蛋白质金属蛋白酶结构域的重组截短形式（重组伊卡米特）在功能和表达方面进行了比较。简而言之，设计了一个由编码伊卡林的序列组成的DNA构建体，并将其克隆到pCAGGS表达载体中，随后在中国仓鼠卵巢（CHO）细胞中进行表达。为了鉴定表达蛋白的酶活性，进行了生物活性测定。比较了重组伊卡林和重组伊卡米特蛋白的凝血时间和表达水平。此外，对用这些蛋白处理的小鼠肝脏进行了组织病理学评估。重组伊卡林和重组伊卡米特表达模式的比较表明，全长伊卡林在真核细胞中的表达至少高2倍。重组伊卡林功能的测定证明该蛋白能够切割凝血酶原并产生凝血酶。重组伊卡林和重组伊卡米特之间的凝血酶原时间（PT）测试结果比较表明，这两种酶的活性存在显著差异，且全长蛋白使血液凝固的时间更短。
