“Self-cleaving” 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella

Xinming Tang,Xianyong Liu,Xun Suo,Mei Qin,Guangwen Yin,Geru Tao,Jingxia Suo

doi:10.1186/s13567-016-0351-z

Abstract

The “self-cleaving” 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more “self-cleaving” 2A sequences. In this study, we introduced a single 2A sequence of porcine teschovirus-1 (P2A) linked to two fluorescent protein genes, the enhanced yellow fluorescent protein (EYFP) gene and the red fluorescent protein (RFP) gene, in a single cassette into transgenic Eimeria tenella (EtER). As expected, we obtained two separated protein molecules rather than a fused protein, although the two molecules were translated from the same mRNA carrying a single “self-cleaving” 2A sequence. Importantly, RFP led by a secretion signal was secreted into parasitophorous vacuoles, while EYFP localized mainly to the nucleus of EtER. Our results demonstrate that the “self-cleaving” 2A sequence actively mediated cleavage of polyproteins in the apicomplexan parasite E. tenella.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0351-z) contains supplementary material, which is available to authorized users.

Highlights

The “self-cleaving” 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more “self-cleaving” 2A sequences
PCR-based analysis using P1/P2 primers revealed that the transfection vector existed in the transgenic parasite genome, and analysis using P3/P4 primers revealed that enhanced yellow fluorescent protein (EYFP)-red fluorescent protein (RFP) was translated by the same mRNA from cDNA (Figures 1A and C)
Expression of multiple polypeptides from a single mRNA is made possible by inclusion of a short viral 2A peptide coding sequence between the polypeptide-encoding transgenes [11, 25]

Summary

Introduction

The “self-cleaving” 2A sequence of picornavirus, which mediates ribosome-skipping events, enables the generation of two or more separate peptide products from one mRNA containing one or more “self-cleaving” 2A sequences. We report a single cassette system which co-expresses two fluorescent proteins, EYFP and RFP (red fluorescent protein), and is cleaved by P2A in the apicomplexan parasite E. tenella. We constructed a single cassette system in which TgDHFR/EYFP-P2A-ssRFP was flanked by the SAG13 promoter and its 3′ UTR (Figure 1A; Additional file 1).

Results

Conclusion