The construction of a duckweed expression and delivery system for grass carp reovirus VP35

Abstract

Haemorrhagic disease caused by grass carp reovirus (GCRV) in grass carp (Ctenopharyngodon idella) is the main viral disease that diminishes the survival of juvenile grass carp. The usage of vaccines, especially oral vaccines, has been considered an effective and convenient way to protect against this disease. However, oral vaccines are easily degraded and digested by digestive enzymes in the intestinal tract. Therefore, a vaccine delivery system that can protect oral vaccines from degradation is a prerequisite for oral vaccines. In this study, the S11 segment (encoding a 35 kDa protein known as VP35) of GCRV was cloned into a binary vector and then transferred into duckweed (Lemna aequinoctialis) to prepare an oral subunit vaccine. Subsequently, Gus staining, Polymerase chain reaction (PCR), Western blotting, and fluorescence observation were used to verify the expression of the S11 segment. The results showed that duckweed calli were successfully induced and could be used for the transformation of Agrobacterium. Afterwards, a plant binary expression vector (pCAMBIA1303-S11) containing GUS and GFP tags was constructed, after which transgenic duckweed lines were obtained. The results of the PCR analysis using RNA or DNA extracted from transgenic duckweed showed that the expression vector pCAMBIA1303-S11 was successfully transferred and replicated in duckweed. After GUS staining, obvious blue signals appeared at the roots, leaf bases, and frond edges of duckweed, and a strong fluorescent signal could be observed under a fluorescence microscope. A Western blot analysis of the protein samples from the transgenic plants demonstrated the expression of a fusion protein with a molecular mass of approximately 50 kDa. Taken together, the duckweed expression system was successfully constructed, and an oral vaccine based on the S11 segment of GCRV was successfully prepared. These results can provide further information for the development of a duckweed-based expression system to produce an edible vaccine for C. idella against GCRV.