Studies On The Transformation Of Fusion Gene VP₇-LK-IFN_a-2b Into Tomato

Rotavirus is the most important pathogen of causing severe diarrhea in young children all over the world, and the disease percentage and mortality are very high in developing countries. Now it is the high time to develop a kind of safe and effective rotavirus vaccine. VP7 is the major outer capsid and is a primary candidate for inclusion in a subunit or recombinant vaccine of rotavirus, Interferon (IFN) is a key cytokine and plays a critical role in inducing the antiviral and antigrowth responses and modulating immune reactions. It has been shown that cytokine could be easily carried to the targeting cells by antigen and give play to role of immunological adjuvant. Through VP7-Lk-IFNa-2b fusion gene was constructed and introduced into Tomato, which provide a new pathway for developing transgenic plant oral vaccine to prevent and treat RV enterities.In this article, VP7-Lk-IFNa-2b fusion gene which size is 1500bp was constructed by recombinant PCR technique. With the aim of being constructed fusion gene, VP7 gene and IFNa-2b gene were connected by seven encoding sequences of myoglobulin heavy chain. Then the constructed fusion gene was introduced into a plant expression vector of pBI121 and a plant expression vector of pBI121/ VP7-Lk-IFNa-2b was constructed, which was transferred into agrobacterium tumefaciens of EHA105. By Agrobacterium-mediated transformation, VP7-Lk-IFNa-2b fusion gene was introduced into tomato. With kanamycin selection, six regenerated tomato plants were obtained, which were proved as be transgenic plants by PCR, PCR-Southern blot. But Southern blot analysis suggested that VP7-Lk-IFNa-2b fusion gene had been integrated into genomes of two PCR/ PCR-Southern blot-positive regenerated tomato plants.SDS-PAGE analysis indicated that six transgenic plants of PCR/ PCR-Southern blot-positive regenerated tomato plants had a better expression at protein levels and a fusion protein with relative molecular mass (Mr) of 55kD, which is accord with theoretic size of VP7-LK-IFNa-2b fusion protein .