In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species.
In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species.
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DOI：
10.1038/s41598-018-32099-6
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发表时间：
2018-09-19
影响因子：
4.6
通讯作者：
Ehsani P
中科院分区：
文献类型：
--
作者：
Amiri M;Jalali-Javaran M;Haddad R;Ehsani P
人组织型纤溶酶原激活物是参与中风后血栓分解的最重要的治疗性蛋白质之一。在1541 bp处发现突变（G514 E），并将突变形式克隆到双元载体pTRAc-ERH中。计算机模拟分析表明，该突变可能对组织型纤溶酶原激活剂酶的活性位点没有显著影响。因此，酶谱分析证实了突变形式及其衍生物的丝氨酸蛋白酶活性。突变形式的表达在有/没有P19基因沉默抑制子共农杆菌注射的情况下在烟草和烟草中得到验证。本萨米亚纳酶联免疫吸附试验结果表明，在无P19存在的情况下，突变体的浓度分别为总可溶性蛋白的0.65%和0.74%，而在有P19存在的情况下，突变体的浓度分别为0.141%和1.36%。benthamiana和N. tabacum。在N.烟草，共农杆菌注射P19具有协同作用，并使突变的组织纤溶酶原激活物的产生增加两倍。然而，在N.在本塞姆那中，P19的存在具有浓度降低五倍的不利影响。此外，结果表明，突变形式及其衍生物的活性高于纯化的商业组织纤溶酶原激活剂。
Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.
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影响因子：
8
作者：
COLOVOS, C;YEATES, TO
通讯作者：
YEATES, TO
影响因子：
3.9
作者：
Ceaglio, N.;Etcheverrigaray, M.;Oggero, A.
通讯作者：
Oggero, A.
影响因子：
2.9
作者：
HEUSSEN, C;DOWDLE, EB
通讯作者：
DOWDLE, EB
影响因子：
17.3
作者：
HOCKNEY, RC
通讯作者：
HOCKNEY, RC
影响因子：
4.8
作者：
ALLEN, S;NAIM, HY;BULLEID, NJ
通讯作者：
BULLEID, NJ