| Noroviruses (NVs) is a pathogen which is resulting in epidemic nonbacterial gastroenteritis in human beings, NVs is a set of virus particles with similar forms and slightly different antigenicity, and belongs to Norovirus in the Human Calicivirus (HuCV). This virus can infect people of all ages and it has a very strong infectivity, and is just following rotavirus. It is ease to break out in the crowded place and the patient’s condition is difficult to control.Currently, there is no specific drug to prevent or cure the infections caused by NVs in clinic yet. In recent years, gastroenteritis leaded by NVs often broke out in several countries in the world on a large scale. The vaccine has become important methods to prevent the infection of the NVs and the virus-like particles (VLPs) have been concerned by several research groups. The research results showed that NVs capsid protein P particles have specific affinity with Histo-blood group atigen (HBGA) of people, and is far stronger than other VLPs. Therefore, it maybe is a good idea to produce P particle edible vaccine to prevent or cure infections and outbreak of NVs.In this study, a PGENE which is encoding P particle protein of the VA387 strain in NVs was designed and synthesized according to tomato codon usage bias, and a DNA sequence which encodes six histidines was fused in 5’end of the PGENE’ to ease for purification of the P particle protein. Afterward, the PGENE with encoding sequences of the 6×his was constructed at E8 promoter downstream to create E8::PGENE, which is a binary vector of the fruit-specific expression in tomato. The explants of tomato hypocotyls was transformed via Agrobacterium tumefaciens -mediated method. Synchronously, a prokaryotic expression plasmid of the pET32a-PGENE was also constructed, and introduced into Escherichia co//(strain BL21). The P particle protein was expressed in E.coli, and the purified P particle protein was obtained. The E.coli-derived P particle protein will be as a important reference for research of the tomato-derived P particle protein.277 tomato plants which is resistant to kanamycin(75 mg/L) was obatained,14 plants of which have been found that the PGENE was successful integrated into their genome via PCR and Southern blotting. The results of RT-PCR and Western blotting demonstrated that the PGEAE has been expressed at levels of transcript and translation, and content of the P particle protein is 0.22% in TSP(total soluble protein). The functional round P particles were successful discovered under TEM(Transmission Electron Microscope). Results of the immunological assay in mouse showed that the bioactive antiserums were obtained through injected P particles which are from both systems of tomato and E.coli. These results will be necessary to express edible NVs vaccine using transgenic tomato. |
