Zaher Mostafa R, El-Husseini Dalia M, El-Husseiny Mohamed H, El Amir Azza M, Hagag Naglaa M, Tammam Reham H
Genome Research Unit, Animal Health Research Institute, Agriculture Research Center (ARC), Giza, 12618, Egypt.
Biotechnology Department, Faculty of Science, Cairo University, Giza, 12613, Egypt.
Biotechnol Lett. 2025 Apr 21;47(3):44. doi: 10.1007/s10529-025-03583-7.
Design and validate flexible constructs for recombinant expression of FMDV serotype O structural proteins of the circulating topotypes using newly designed degenerate primers. The designed degenerate primers targeting diverse topotypes enabled the successful amplification of VP0, VP1, and VP3 genes. Integration of the essential transcriptional and translational regulatory elements including T7 promoter, leader g10 sequence, and T7 terminator, as well as ribosome binding site (RBS), start and stop codons, respectively via overlap extension PCR empowered efficient expression of these proteins in E. coli. Cloned constructs expressed the target proteins of expected molecular weights: VP0 (34 kDa), VP1 (24 kDa), and VP3 (22 kDa). SDS-PAGE and Western blotting confirmed high protein yield and purity. This platform demonstrated adaptability for diagnostic and vaccine development applications. The workflow offers a robust tool for producing FMDV structural proteins concerning the circulating strains attempting to improve control measures including diagnosis and vaccinations.
使用新设计的简并引物设计并验证用于循环拓扑型口蹄疫病毒O型结构蛋白重组表达的灵活构建体。针对不同拓扑型设计的简并引物能够成功扩增VP0、VP1和VP3基因。通过重叠延伸PCR分别整合包括T7启动子、前导g10序列和T7终止子以及核糖体结合位点（RBS）、起始和终止密码子在内的必需转录和翻译调控元件，可使这些蛋白在大肠杆菌中高效表达。克隆的构建体表达出预期分子量的目标蛋白：VP0（34 kDa）、VP1（24 kDa）和VP3（22 kDa）。SDS-PAGE和蛋白质免疫印迹证实了高蛋白质产量和纯度。该平台展示了在诊断和疫苗开发应用中的适应性。该工作流程为生产与循环毒株相关的口蹄疫病毒结构蛋白提供了一个强大的工具，有助于改进包括诊断和疫苗接种在内的控制措施。
