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Expression of dengue-3 premembrane and envelope polyprotein in lettuce chloroplasts

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Abstract

Dengue is an acute febrile viral disease with >100 million infections occurring each year and more than half of the world population is at risk. Global resurgence of dengue in many urban centers of the tropics is a major concern. Therefore, development of a successful vaccine is urgently needed that is economical and provide long-lasting protection from dengue virus infections. In this manuscript, we report expression of dengue-3 serotype polyprotein (prM/E) consisting of part of capsid, complete premembrane (prM) and truncated envelope (E) protein in an edible crop lettuce. The dengue sequence was controlled by endogenous Lactuca sativa psbA regulatory elements. PCR and Southern blot analysis confirmed transgene integration into the lettuce chloroplast genome via homologous recombination at the trnI/trnA intergenic spacer region. Western blot analysis showed expression of polyprotein prM/E in different forms as monomers (~65 kDa) or possibly heterodimers (~130 kDa) or multimers. Multimers were solubilized into monomers using guanidine hydrochloride. Transplastomic lettuce plants expressing dengue prM/E vaccine antigens grew normally and transgenes were inherited in the T1 progeny without any segregation. Transmission electron microscopy showed the presence of virus-like particles of ~20 nm diameter in chloroplast extracts of transplastomic lettuce expressing prM/E proteins, but not in untransformed plants. The prM/E antigens expressed in lettuce chloroplasts should offer a potential source for investigating an oral Dengue vaccine.

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Acknowledgments

The investigations reported in this study were supported in part by grants from USDA 3611-21000-021-02S and NIH R01 GM 63879-08 to HD. We thank AMPAC electron microscope facility at the University of Central Florida and Dr. Qi Zhang for her assistance in transmission electron microscopy studies. We thank Prof. Rofina Yasmin Othman and Ms. Lin Shina for providing the plasmid pDEN3 and Dr. Johari Bin Mohd Ali (Institute of Biological Sciences and University of Malaya, Malaysia) for technical assistance.

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  1. Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida, 336 Biomolecular Science Building, Orlando, FL, 32816-2364, USA

    Anderson Paul Kanagaraj, Dheeraj Verma & Henry Daniell

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  1. Anderson Paul Kanagaraj
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Correspondence to Henry Daniell.

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Kanagaraj, A.P., Verma, D. & Daniell, H. Expression of dengue-3 premembrane and envelope polyprotein in lettuce chloroplasts. Plant Mol Biol 76, 323–333 (2011). https://doi.org/10.1007/s11103-011-9766-0

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