Гилева И П, Якубицкий С Н, Колосова И В, Щелкунов С Н
Государственный научный центр вирусологии и биотехнологии «Вектор» Роспотребнадзора, р. п. Кольцово, Новосибирская область, Россия.
Государственный научный центр вирусологии и биотехнологии «Вектор» Роспотребнадзора, р. п. Кольцово, Новосибирская область, Россия Федеральный исследовательский центр Институт цитологии и генетики Сибирского отделения Российской академии наук, Новосибирск, Россия.
Vavilovskii Zhurnal Genet Selektsii. 2020 May;24(3):239-243. doi: 10.18699/VJ20.616.
Tumor necrosis factor (TNF) is one among the key cytokines that mediate the immune system to protect humans against viral infections. Throughout evolution, anthropogenic Variola virus (VARV) has developed effective mechanisms to overcome human defense reactions. The viral genome encodes soluble proteins imitating the structure of cellular cytokine receptors. These proteins compete with cellular receptors for cytokine binding, thus blocking the antiviral immune response. In particular, the G2R gene of VARV encodes the TNF decoy receptor, VARV-CrmB protein. This protein consists of N-ended TNF-biding (TNF-BD) and C-ended chemokine binding (Ch-BD) domains. Recombinant VARV-CrmB protein has been produced in insect cells using molecular cloning methods and its TNF neutralizing activity has been shown in vitro and in vivo. To decrease the immunogenicity of this protein, a recombinant plasmid coding for shortened TNF-BD protein of VARV in Escherichia coli cells has been constructed. Using the method of immobilized metal affinity chromatography, recombinant TNF-BD protein corresponding to the TNF-biding domain of VARV-CrmB protein was purified from E. coli cells. The therapeutic potential of TNF-BD was studied using an experimental model of LPS-induced septic shock. After septic shock induction, several doses of recombinant TNF-BD were injected and the mortality of experimental animals was observed during 7 days. All mice not injected with TNF-BD had been dead by day 3 of the experiment, but 30, 40 and 60 % of the experimental animals, who received different TNF-BD doses, survived in a dose-dependent manner. Data obtained demonstrate that recombinant TNF-BD protein is pharmacologically active in the experimental model of LPS-induced septic shock.
肿瘤坏死因子（TNF）是介导免疫系统保护人类抵御病毒感染的关键细胞因子之一。在整个进化过程中，人为的天花病毒（VARV）已形成有效的机制来克服人类的防御反应。病毒基因组编码模仿细胞因子受体结构的可溶性蛋白质。这些蛋白质与细胞受体竞争细胞因子结合，从而阻断抗病毒免疫反应。特别是，VARV的G2R基因编码TNF诱饵受体，即VARV-CrmB蛋白。该蛋白由N端的TNF结合（TNF-BD）和C端的趋化因子结合（Ch-BD）结构域组成。重组VARV-CrmB蛋白已通过分子克隆方法在昆虫细胞中产生，并且其TNF中和活性已在体外和体内得到证实。为了降低该蛋白的免疫原性，已构建了在大肠杆菌细胞中编码VARV缩短的TNF-BD蛋白的重组质粒。使用固定化金属亲和色谱法，从大肠杆菌细胞中纯化了与VARV-CrmB蛋白的TNF结合结构域相对应的重组TNF-BD蛋白。使用LPS诱导的脓毒症休克实验模型研究了TNF-BD的治疗潜力。在诱导脓毒症休克后，注射了几剂重组TNF-BD，并在7天内观察实验动物的死亡率。所有未注射TNF-BD的小鼠在实验第3天死亡，但接受不同TNF-BD剂量的实验动物中，分别有30%、40%和60%以剂量依赖方式存活。获得的数据表明，重组TNF-BD蛋白在LPS诱导的脓毒症休克实验模型中具有药理活性。
