Construction Of Plant-Based Expression Vector Of Transgenic Plant Vaccine Against The Grass Carp Hemorrhage

Grass carp(Ctenopharyngodon idellus), is a most popular commercial fish species in China. Grass Carp Hemorrhage is one of the most serious diseases of grass carp, causing more than 90% mortality and huge economic loss to the aquaculture industry. At present, no effective ways for prevention and treatment of the disease are available.Transgenic plant-derived vaccine is a kind of new vaccine that combines the technology of molecular cloning, plant gene engineering and immunology. It is prepared by constructing the pant-based expression vector and deliverring foreign genes into plants and then elicits the special immune response in the body of human or animal through oral approach. Compared with the traditional vaccines, transgenic plant-derived vaccine is cheaper, safer and more efficacious.Recently, more and more antigen proteins have been expressed in plants and tested in animals or human.In this study, plant-based expression vector containing the Grass carp reovirus VP6 antigen protein gene was constructed and the calli of ryegrass was induced. The present study has made a solid base for the innovation of edible transgenic plant vaccine against grass carp hemorrhage caused by reovirus through oral immunization. The study was carried out from the following aspects.1.The coding gene of grass carp reovirus (GCRV) VP6 antigen protein was amplified by RT-PCR from the viral RNA genome extracted in virus infected Ctenopharyngodon idellus Kidney (CIK) cells with primers based on the gene sequence in GeneBank (AF403394).The 1.3kb PCR product was ligated to pCR2.1 vector, and after identified with enzyme digestion, PCR detection and sequencing, the 1.3kb cording sequence of GCRV VP6 gene was cloned into the plant-based expression vector pCAMBIA1302, which had a green fluorescent protein (GFP) gene insert. Thus, a fusion-expression vector of grass carp reovirus VP6 gene and GFP gene was constructed.2.Specific primer pairs for amplifying the GCRV VP6 gene and the Escherichia Coli LTB gene were designed and synthesized based on the sequences of GCRV VP6 gene and Escherichia Coli LTB gene posted in GenBank. GCRV genome RNA was extracted from the virus infected CIK cells and the GCRV VP6 coding region was amplified by RT-PCR; The LTB coding region was amplified from Escherichia Coli H44815 genome.The PCR products were cloned into pCR2.1 vector respectively and then sequenced and subcloned into vector pCAMBIA 1302, which contained a green fluorescence protein gene marker. Fusion expression vector of LTB, grass carp reovirus VP6 gene and green fluorescence protein gene vector was successfully constructed.3.The calli induction, subculture, differentiation and plant regeneration of ryegrass were conducted in this study. The embryonic calli can be used to tissue culture.