Assembling of gene sequences encoding epitopic regions of foot and mouth disease virus non-structural proteins using one-step OE-PCR

Document Type : Research Paper


1 Department of cell and molecular Biology, Kharazmi University, Karaj

2 Department of Anaerobic Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Karaj

3 Razi Vaccine and Serum Research Institute, Karaj

4 Department of Anaerobic Bacterial Vaccine Research and Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran


Foot-and-mouth disease (FMD) is an acute highly contagious viral disease in susceptible cloven-hoofed animals, which causes extensive economic loss in livestock industry. Recently, diagnostic methods have been developed to detect antibodies against non-structural proteins of this virus. Non-structural proteins 3ABC and 3D that provide linear B cells epitopes allow effective detection of virus-infected animals. In this study, immuno-dominant epitopes of 3ABD proteins were considered for the synthesis of a new gene sequence and assembling of 3AB and 3D fragments.
Materials and Methods
Overlap extension-polymerase chain reaction (OE-PCR) was used to synthesize 3ABD antigenic sequences. Viral RNA was isolate from BHK-infected cells and reverse transcribed. In order to remove 654 nucleotides of the middle 3C genomic sequence, a pair of internal primers with 29 overlapping nucleotides and two external primers were designed. The 3AB and 3D fragments were each amplified in separate PCR reactions and purified from the gel after electrophoresis. These fragments were used as templates in a one-step OE-PCR for the synthesis of 3ABD sequence by external primers and Pfu DNA polymerase.
The results showed amplification of 3AB (418 bp) and 3D (557 bp) fragments with overlapping sequences at the end of 3' and 5', respectively. The OE-PCR experiment resulted in amplification of multiple fragments, and the 3ABD fragment with the desired length (946 bp) was confirmed after sequencing. Only by considering 29 homologous nucleotides in the design of overlapping primers and a high-fidelity, highly accurate DNA polymerase, two epitopic regions of non-structural proteins of FMD virus (3'-3AB and 5'-3D) were linked together.
The new gene sequence synthesized by a one-step OE-PCR technique has the antigenic sites of 3ABD non-structural proteins of FMD virus, obviates the need for expensive peptide synthesis, and can be a proper alternative for production of 3ABD recombinant protein for application in detection of FMD by Enzyme Linked Immunosorbent Assay.


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