Puckette Michael, Clark Benjamin A, Smith Justin D, Turecek Traci, Martel Erica, Gabbert Lindsay, Pisano Melia, Hurtle William, Pacheco Juan M, Barrera José, Neilan John G, Rasmussen Max
U.S. Department of Homeland Security Science and Technology Directorate, Plum Island Animal Disease Center, Greenport, New York, USA
Leidos, Inc., Plum Island Animal Disease Center, Greenport, New York, USA.
J Virol. 2017 Oct 27;91(22). doi: 10.1128/JVI.00924-17. Print 2017 Nov 15.
The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria. The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production.
口蹄疫病毒（FMDV）在80多个国家感染家畜，限制了粮食生产和全球贸易。口蹄疫（FMD）疫苗的生产需要在胞质中表达FMDV 3C蛋白酶，以将P1多蛋白切割成成熟的衣壳蛋白，但FMDV 3C蛋白酶对宿主细胞有毒性。为了鉴定毒性较小的FMDV 3C蛋白酶同工型，我们使用荧光素酶报告系统筛选了3C突变体，以比较与野生型3C相比转基因产量的增加情况。新的点突变3C（L127P）提高了表达P1-3C转基因的哺乳动物和细菌细胞中重组FMDV亚基蛋白的产量，并保留了处理多种FMDV血清型P1多蛋白的能力。3C（L127P）突变体在哺乳动物和细菌细胞中产生了口蹄疫样颗粒的晶体阵列，这可能为在细菌中快速、廉价地生产口蹄疫疫苗提供一种实用方法。突变的FMDV 3C蛋白酶L127P显著提高了重组FMDV亚基抗原的产量，并在哺乳动物和细菌细胞中产生病毒样颗粒。L127P突变代表了口蹄疫疫苗经济生产的一项新进展。
